Dissociation constant value (pKa) is key parameter for predicting the extent of the ionization of a drug molecule at different pH. In this study, pKa 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 pKa values, the aqueous pKa 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.