To establish a HPLC method for related substances determination of esomeprazole magnesium enteric coated capsules.The determination was carried out on a column of Welch C18 (150 × 4.6 mm, 5μm).The mobile phase consisted of a mixture of acetonitrile, phosphate buffer (pH 7.6) and water by gradient elution, with a flow rate of 1 mL/min at 30°C.The detection wavelength was 302 nm.The calibration curves were linear in the range of 0.08-8.0μg/mL (r = 0.9999) for esomeprazole magnesium, and 0.224-22.4μg/mL (r = 1) for omeprazole sulfone.The average recovery (n = 9) of omeprazole sulfone was 99.5%, RSD was 1.6%.The contents of omeprazole sulfone, any other individual impurity and total impurities were acceptable for all 4 batches.This method shows good precision and high accuracy, and can be applied to the related substances determination in esomeprazole magnesium enteric-coated capsules.