An optimization methodology is introduced for investigating the retention behavior and the separation factor of met formin, gliclazide (I. S.) and glibenclamide. This investigation has been focused on studying the influence of pH value of the mobile phase, concentration of acetonitrile and column temperature, which affect a complete separation of the chromatographic peaks of these compounds. The significant factors were optimized using full factorial design. Retention factor and separation factor were chosen as dependent variable. Optimum RP-LC chromatographic conditions for the separation of metformin, glibenclamide and gliclazide were obtained using X Terra column (150 mm x 4.6 mm I. D., 5 mu m). The results show that the percentage of acetonitrile are the most important to investigate and (s)(s)pH of the mobile phase and column temperature do not significantly affect the experimental results. The procedure was validated for linearity, accuracy, precision and recovery. Quantitation was accomplished using internal standard method.