Back ground: Second-line HIV treatment failure has become increasing worldwide, mainly in sub- Sahara Africa including Ethiopia. Even though the problem becomes increasing, inadequate information was available about its magnitude and predictors in the current study area. Objective: To assess the predictors of second line Anti-Retroviral Treatment virological failure among second line ART users. Method and materials: Institutional based unmatched case control study design was conducted from first September 2021 to December last 2021 at Felege Hiowt and University of Gondar Comprehensive Specialized Hospitals; Amhara region, North West Ethiopia. A total of 216 patients (60 cases and 156 controls) were recruited by Simple random sampling technique with 1:3 cases-to-controls ratio. Patients who had two viral load results >1000 copies/ml within a 3-month interval after taking ART drugs for at least 6 months were cases whereas ≤1,000 copies/ mL were controls. The sample size was calculated by using Epi-Info version 7.2.4. Structured questionnaires and check lists were used to gather the required information from patient registration book/ card/ review and face to face interviews. SPSS version 26 was used to summarize the findings. In bivariate logistic regression model, Variables with two-tailed P- value ≤ 0.25 at 95% confidence interval were transferred in to multivariate binary logistic regression mode and P value at ≤ 0.05 was set as statistically significant. Results: Out of 216 patients recruited, 212 were participated with a response rate of 98.2%. Among the participants, 117 (55.2%) were males and 187 (88.2%) were urban dwellers. 208 (98.1%) of the respondents had age > 24 years, 73 (34.4%) had elementary level of education, 72(34%) had poor ART adherence and 112(52.8) did not disclose their HIV status. Likewise, Most of the patients 147(69.37) didn’t used condom. The Predictors were; not disclosing HIV status (AOR=3.4, 95% CI: 1.52 – 7.79), poor adherence level (AOR=5.27, 95% CI: 2.2 - 12.5), not using condom (AOR=4.47, 95% CI: 1.63 – 12.2) and high Viral load (>1000 copes/ml) when switched to second line ART (AOR=3.56, 95% CI: 1.5 - 8). Conclusion and recommendations: The Predictors of second line Anti-Retroviral Treatment virological failure were non-disclosure, poor adherence, not using condom and high Viral load (>1000 copes/ml) at switched to second line ART. Disclosing their HIV status, using condom and improving their adherence level for patients and counselling about the importance of disclosure and good adherence for health care providers are crucial. Key word: ART Virological failure, Felege Hiwot Hospital, University of Gondar Hospital.