Dissociation constant value (pK_a) is key parameter for predicting the extent of the ionization of a drug molecule at different pH. In this study, pK_a values of famotidine, ranitidine and nizatidine in different percentages of acetonitrile-water binary mixtures (10%, 15%, and 20%, v/v) were determined from the mobile phase pH dependence of retention factor with reverse phase liquid chromatographic method (RPLC). From calculated pK_a values, the aqueous pK_a values of studied compounds were estimated by mole fraction of acetonitrile. Moreover, the correlation established between retention factor and the pH of the water-acetonitrile mobile phase was used to determination of optimum separation condition. In order to validate the optimized condition, famotidine, ranitidine and nizatidine were studied in their dosage forms. A X-Terra C-18 reverse-phase column (250x4.6mm I.D., 5 μm particles) was preferred to carry out the developed method. Mobile phase of acetonit- rile-methanol-water (10:6:84, phosphate buffer pH: 6.5) and diode array detection system at wavelengths 210 and 320 nm were used as separation conditions. Under studied conditions detection limits of 0.5678 μg/mL for famotidine, 0.3100 μg/mL for ranitidine and 1.3144 μg/mL for nizatidine were found. The parameters, linearity, precision, accuracy, limit of detection, and limit of quantitation were studied according to U.S. Pharmacopoeia.