A novel gradient reversed-phase High performance liquid chromatographic method has been developed for quantitative determination sulfide impurity of esomeprazole. The method display quantification potential related substances Sulfide (Impurity C) of Esomeprazole. Mobile phase A, B consists of 0.01M Disodium hydrogen phosphate: acetonitrile (80:20, v/v) and (30:70, v/v) respectively, pH-7.6 with orthophosporic acid, which were run as per the optimized gradient program. Zorbax SB C-8, (150cm×4.6 mm, 5μm) was used as the stationary phase. 1.0 mL per minute was employed as the flow rate and injection volume was kept as 40 μL. Detection wavelength was kept as 280 nm. The method was validated as per ICH Q2 (R1) guidelines &confirmed a good performance with respect to specificity, linearity, sensitivity, accuracy, precision, selectivity. So the proposed method can be used in routine quality control laboratories.