Objective: This research aimed to develop sensitive, simple, and selective methods for determination of esomeprazole in human plasma using lansoprazole as internal standard by high performance liquid chromatography. Method: The analytical separation was performed on C-18 column (Waters, Sunfire™ 5 μm; 250 × 4.6 mm), column temperature 40°C. The mobile phase used acetonitrile - phosphate buffer pH 7.6 (40: 60% v/v) with a flow rate of 1.00 mL/min; and photodiode array detector at a wavelength of 300 nm. Esomeprazole and lansoprazole were extracted by liquid-liquid extraction using dichloromethane. Results: The method had a chromatographic run time of 10 min and linear calibration curve over the range of 5.0-450 ng/mL with a correlation coefficient (r) of 0.999 or better. The sensitivity, specificity, linearity, precision and accuracy, recovery and stability were validated for esomeprazole in human plasma. Conclusion: The results of validation fulfilled the acceptance criteria of validation method based on EMEA Bioanalytical Guideline 2011.