β-lactam antibiotics are used to treat bacterial infections. Despite a high level of clinical success, they have encountered a serious resistance that demands a high dose regimen and a new pharmacokinetic combination. This requires continuous monitoring of their level in pharmaceutical and biological samples. In this study, electrochemical sensors were developed for the determination of selected β-lactam drugs (Amoxicillin (AMX), cefadroxil (CFL), procaine penicillin G (ProPenG), and cephalexin (CLN)) in pharmaceutical formulations and biological fluid samples. The sensors were developed by modifying glassy carbon electrode (GCE) using novel metal complexes, (aquachlorobis(1,10-phenanthroline)copper(II) iodidemonohydrate, and diresorcinate-1,10-phenanthrolinecobalt(II)), as well as conducting polymers (4-amino-3-hydroxynaphthalene sulfonic acid, alizarin, and resorcinol). Cyclic voltammetry (CV) and electron impedance spectroscopy (EIS) results revealed modification of the electrode surface by a conductive, and electroactive polymer film leading to an increased effective electrode surface area and conductivity. The appearance of an irreversible oxidative peak at much-reduced potential with five folds current enhancement at poly(ACP2CuIH)/GCE showed the catalytic effect of the modifier towards oxidation of AMX. Relative to the peak of CFL on the bare electrode, an irreversible oxidative peak on poly(Alz)/GCE with four folds of current was observed at a reduced potential verifying the improvement of conductivity and effective surface area of the electrode surface. In contrast to the unmodified electrode, an irreversible oxidative peak at poly(DHRPCo)/GCE with six folds of current enhancement at lower potential was observed, confirming the electrocatalytic effect of the polymer-modified electrode. Under optimized film thickness, solution pH and pulse parameters, linear concentration range, LoD, detected levels of tablet brands, and %recovery results in pharmaceutical and biological samples were summarized in the table below. The result showed excellent agreement between the detected amount and company label, excellent spike recovery results with good interference recovery less than 4.9% error, wider LDR, and lowest LoD than most of the previously reported methods along with its excellent accuracy and selectivity; validated the potential applicability of the present methods for determination of AMX, CFL, ProPenG and CLN in real samples. x Table: Summary of LDR, LoD, % recovery, and % detected of each analyte with their sensors Modifier Analyte LDR (µM) LoD (µM) Detected % %Recovery poly(ACP2CuIH) AMX 0.5–100.0 0.013 95.2–101.8% 98.8–100.6% poly(AHNSA/GCE 10.0–150.0 0.0099–0.010 97.84–100.8% 99.6–100.5% poly(Alz)/GCE CFL 0.1–100.0 0.0081 99.0–99.5 99.5–100.5% poly(DHRPCo)/GCE ProPenG 0.1–200.0 0.0049 96.1–101.3% 98.0–106.5% poly(reso) CLN 0.1 – 300.0 0.00312 91.00–103.6% 99.0–100.6%, CFL 0.5 – 300.0 0.0087 97.7–98.8% 97.1–100.6% For better certainty about the nature of the surfaces of the developed sensors, we recommended further characterization using scanning electron microscopy (SEM), atomic force microscopy (AFM), and x-ray photoelectron spectroscopy (XRPES).