Evaluation of protein binding based in vitro interaction between ticagrelor and pantoprazole was carried out by a reversed phase HPLC method. An efficient RP-HPLC method was developed for the simultaneous estimation of ticagrelor and pantoprazole and applied to evaluate protein binding based interaction of two drugs using equilibrium dialysis method. The amount of free and bound drugs was estimated by using reverse phase chromatographic method with photo diode array detection. Shimadzu C18 column (250 mm × 4 mm, 5 μ) was used to separate ticagrelor from pantoprazole. The mobile phase used was 10 mM potassium dihydrogen orthophosphate: methanol (20:80 %v/v) at a flow rate of 1 ml/min. The drugs were detected at wavelength of 254 nm and peak areas were integrated. The retention time for pantoprazole was 3.1 min and ticagrelor was 7.8 min. The developed method was validated and successfully applied for the evaluation of protein binding based in-vitro interaction of ticagrelor and pantoprazole. The protein binding study results have shown that there is a significant change in the binding of two drugs in presence of each other and hence might affect the effectiveness of them and lead to adverse effects.