Objective: A novel reverse-phase high-performance liquid chromatography (RP-HPLC) method for estimation of lamivudine and zidovudine by using an experimental design approach applying the response surface technique was developed and validated using a C18 column and its application in marketed formulation. Multivariate optimization of experimental conditions was achieved using experimental design employing organic content in the mobile phase, pH, and flow rate of the mobile phase as three independent variables. The aim of this study was to apply response surface methodology and to study the effect of the independent variables on the separation and estimation of both drugs by the RP-HPLC method using a faced central composite experimental design. Materials and Methods: Derringer's desirability function was used to optimize the retention period of the last eluting peak and peak symmetry, and it was discovered that the optimal conditions were potassium dihydrogen phosphate buffer and acetonitrile in an isocratic ratio of 55:45 w/v, pH 3.5, and 0.4 ml/min flow rate. Baseline separation of both medications with good resolution and a run time of more than 7 min was accomplished using this ideal condition. Results: For lamivudine and zidovudine, a linear response was seen over the concentration range of 2–12 and 4–24 μg/mL, respectively. Lamivudine's limit of detection and limit of quantitation were determined to be 3.08 and 9.18 μg/mL, respectively, whereas zidovudine's values were 3.24 and 10.17 μg/mL. According to the ICH guidelines acceptance criteria for linearity, accuracy, precision, specificity, and robustness, the method was successfully validated.
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