A robust method, for the chromatographic separation of three antidiabetic drugs viz metformin, pioglitazone and glimepiride using an isocratic reversed phase high-performance liquid chromatographic (HPLC) system having ultraviolet detection at 254 nm is presented in this paper. The method was developed in human serum and dosage formulation with high-quality chromatographic separation between the drug peaks by using a stainless steel analytical column Nucleosil, C₁₈ (10 micron, 25 × 0.46 cm). The system was operated at room temperature using a mobile phase consisting of acetonitrile, phosphate buffer (pH 4.3) in the ratio of 60 : 40 v/v at a flow rate of 1 mL min⁻¹. The parametric statistics, i.e., correlation coefficient of 0.999 was assessed for all the drugs having linearity over the tested concentration range (10 to 10 000 ng mL⁻¹) in human serum. The accuracy and the relative standard deviations of samples for six replicate measurements were not less than 97% and greater than 2%, respectively. The proposed method was validated for selectivity, linearity, accuracy, and precision according to the International Conference on Harmonization (ICH). The method is applicable for the quality control of the mentioned drugs in raw material, bulk drug, and pharmaceutical formulations as well as in human serum.