The prime aim of the current work is to develop and validate a novel, sensitive, reverse phase High Performance Liquid Chromatography (RP-HPLC) technique for the estimation of Glibenclamide in dosage form. Chromatographic separation was achieved on a Chromosil column, (150mm×4.6mm x5µ) using an isocratic method with mobile phase composed of Potassium di-hydrogen phosphate buffer (pH 4.5): Acetonitrile in the ratio 60:40 v/v. The flow rate was1 ml/min, temperature of the column was maintained at ambient and detection was made at 233 nm. The run time was 12 min. The developed method was validated according to the International Conference on Harmonization (ICH) guidelines with respect to linearity, accuracy, precision, specificity and robustness. The developed method was linear for Glibenclamide from 10 – 50 μg/ml and the linear regression obtained was > 0.999. Precision, evaluated by intra-and inter-day assays had relative standard deviation (R.S.D) values within 1.5 %. Recovery data were in the range 98.2% to 100.9% with R.S.D. values < 1.5 %. The method is precise, accurate, linear, robust and fast. The short retention time allows the analysis of a large number of samples in a short period of time and, therefore, should be cost- effective for routine Quality Control in the pharmaceutical industry.