A simple and sensitive high performance liquid chromatography method was developed for determination of glibenclamide in presence of its impurities in pharmaceutical dosage form and human plasma. Instrumentation of the method was very simple and used the mixture of acetonitrile: water (60:40, v/v) as the mobile phase. Separation was carried out on a BDS. Hypersil C8 (5 um, 250 × 4.6 mm) column. Effects of composition of mobile phase in addition to flow rate and detection wave length were studied. Calibration was obeyed in the range of 20-100 μg/ml of glibenclamide. The method was validated according to ICH parameters.