Background: Despite the fact that a substantial number of HIV patients have received antiretroviral therapy, the proportion of HIV patients who are receiving second-line antiretroviral therapy is becoming a growing public health concern. In Ethiopia, a large number of HIV patients experienced first-line treatment failure and were switched to second-line antiretroviral therapy. Unlike patients on first-line antiretroviral therapy, however, little is known about medication adherence, depression, and treatment outcomes after the commencement of second-line therapy. Hence, addressing those information gaps will contribute to prolonging the use of second-line antiretroviral therapy, prevention of further treatment failure, reduction of HIV transmission, and improvement of overall HIV care and treatment. Objectives: This study aimed to assess antiretroviral medication adherence, depression, and treatment outcomes among adult HIV patients who were receiving second-line antiretroviral therapy in the eastern Amhara Region, northeast Ethiopia. Methods: We employed both quantitative and qualitative methodology. A retrospective cohort and a multi-center institution-based cross-sectional study were used for the quantitative approach. A phenomenological approach, on the other hand, was used for the qualitative component. A retrospective cohort study was conducted on 642 adult HIV patients who were receiving secondline antiretroviral therapy from October 2016 to November 2019 in order to assess viral resuppression, attrition to care, treatment failure, and death. Cox regression model was used to identify factors associated with such outcomes. Furthermore, a multi-center, institution-based cross-sectional study was also conducted on 714 adult HIV patients on second-line therapy to assess patient antiretroviral medication adherence, depression, and health-related quality of life. Participants were selected via a systematic random sampling method in twenty public health facilities from December 2020 to February 2021. Medication adherence was measured using the six-item simplified medication adherence tool. The nine-item patient health questionnaire and the 31-item WHOQOL-HIV BREF instruments were used to assess depression and health-related quality of life, respectively. Data were collected in a personal interview as well as document reviews. A multi-level binary logistic regression was used to uncover individual and facility-level factors associated with second-line antiretroviral medication adherence. Structural equation modeling, on the other hand, was employed to identify factors associated with depression and health-related quality of life. Statistical significance was declared at a P-value less than 0.05, and the effect sizes were presented using 95% CI. A phenomenological-qualitative approach was also employed to describe the lived experience of HIV patients on second-line medication adherence in order to complement the quantitative findings. Eleven participants were purposefully interviewed, and a thematic analysis was conducted to identify key themes of medication adherence. Results Medication adherence and associated factors The magnitude of medication adherence among HIV patients on second-line antiretroviral therapy was 69.5% (95% CI: 65.9-72.7%). Medication adherence was positively associated with the use of adherence reminder tools [Adjusted odds ratio, AOR = 3.37, (95% CI: 2.03–5.62)], social support [AOR = 1.11, (95% CI: 1.02–1.23)], and not having clinical depression [AOR = 3.19, (95% CI: 1.93–5.27) as compared with their counterparts. Those results were supported by the qualitative findings. At the facility level, the number of adherence counselors [AOR = 1.20, (95% CI: 1.04–1.40)], teamwork for enhanced adherence support [AOR = 1.82, (95% CI: 1.01–3.42)], and caseloads at ART clinic were all significantly correlated with medication adherence. Depression and associated factors Clinical depression was reported in 27.7% of adult HIV patients on second-line therapy [95% CI: 24.7 – 31.1%]. Social support has a direct [𝛽̂ = - 0.9, (95% CI: -1.11 to - 0.69)] and indirect [𝛽̂ = -0.22, (95% CI: - 0.31 to - 0.13)] negative effect on depression. Perceived stigma was a mediator variable and significantly associated with depression [𝛽̂ = 0.40, (95% CI: 0.23 – 0.57)]. Chronic co-morbid illness [𝛽̂ = 0.49, (95% CI: 0.35 – 0.63)], high viremia [𝛽̂ = 0.17, (95% CI: 0.08 – 0.26], moderate and high-risk substance use [𝛽̂ = 0.29, (95% CI: 0.18 – 0.39)], and not-workable functional status [𝛽̂ = 0.2, (95% CI: 0.1 – 0.31)] were positively correlated with depression. Health-related quality of life and associated factors The mean (+SD) score of the overall summary health-related quality of life was 60+13. The spiritual (70 + 20) and environmental (52 + 17) domains had the highest and lowest scores of health-related quality of life, respectively. Social support has a direct and indirect positive effect on psychological and spiritual aspects of health-related quality of life. In contrast, depression and stigma were negatively associated with the psychological, spiritual, and social relationship domains. Of the clinical characteristics, being in a non-workable functional status, advanced clinical stage, high viremia, and having chronic co-morbid illness were variables negatively associated with at least one domain of health-related quality of life. Age was negatively correlated with the physical domain. Not being educated and living in rural areas were also negatively correlated to the environmental domains of health-related quality of life. Viral re-suppression and attrition to care Out of 642 participants, 19 (3%), 44 (6.9%), 70 (10.9%), and 509 (79.3%) were lost to follow up, died, transferred out, and alive on care, respectively. Similarly, 82.4% (95% CI: 79.2–85.1%) of participants had achieved viral re-suppression, with a 96 per 100-person-year rate of resuppression. Patients who switched timely to second-line therapy were at a higher rate of viral re-suppression than delayed patients [Adjusted Hazard Rate, AHR = 1.43 (95% CI: 1.17–1.74)]. Not having a history of drug substitution [AHR = 1.25 (95% CI: 1.02-1.52)], as well as TB co-infection [AHR = 0.67 (95% CI: 0.49–0.91)], were all associated with viral re-suppression. Attrition to care was 11% (95% CI: 8.7-13.9%). Not-workable functional status [AHR= 2.61 (95% CI: 1.40-4.87], high viremia [AHR= 6.87 (95% CI: 3.86-12.23)], CD4 cell count < = 450 cells/mm3 [AHR= 2.61 (95% CI: 1.40-4.87] were positively associated with attrition to care. Second-line treatment failure and death During the follow-up period, 39 (6.87%, 95% CI: 5–9.2%) of 568 patients had second-line treatment failure, with a 4.07 per 100 person-year rate of failure. Treatment failure was associated with TB co-infection [Rate ratio, RR = 2.57 (95% CI: 1.25–5.25)], poor medication adherence [RR = 2.29 (95% CI: 1.09–4.78)], and late treatment switching [RR = 5.89 (95% CI: 1.36–25.54)]. Similarly, 44 (6.85%, 95% CI: 5–9%) of 642 participants died, with a 4.5 per 100 person-year rate of death. Being in advanced clinical condition [Sub distribution Hazard ratio, SHR = 2.49 (95% CI: 1.31- 4.74)], having poor medication adherence [SHR= 2.65 (95% CI: 1.31- 4.74)], and having high viremia were all positively associated with an increased risk of death. Conclusions A large proportion of adult HIV patients on second-line therapy had adherence problems. Both facility and individual-level factors were linked with medication adherence. Similarly, the magnitude of clinical depression in such patients was also high. Social support was the most important determinant factor associated with depression. The treatment outcomes in adult HIV patients after the commencement of second-line therapy were not as expected. Poor clinical conditions and social-behavioral outcomes were linked with poor treatment outcomes. Recommendations Individual and system-level interventions should be targeted based on the identified factors to avert the multi-dimensional consequences of the above issue. Patient-centered monitoring and interventions should be strengthened based on the identified factors, besides treatment switching. Furthermore, new and initiated differentiated models of service delivery (DSD) should be customized and implemented, including community-based ART delivery modalities while strengthening the existing standard facility-based HIV care modalities and clinical mentoring. Keywords: Second-line medication adherence, Depression, Health-related quality of life Viral Re-suppression, Second-line treatment failure, HIV care, and treatmen