Objective: To establish a method for determination assay of empagliflozin and related substance. Methods：HPLC method was adopted. The determination was carried out on Waters symmetry C18 column（250 mm×4.6 mm，5 μm）. The mobile phase A was 0.01%（V/V） trifluoroacetic acid solution，the mobile phase B was acetonitrile，linearity gradient elution was as follows：0~10 min，mobile B 32%；10~50min，mobile B 32%~95%， at a flow rate of 1.0 mL/min. The detective wavelength was set at 225 nm， and the column temperature was at 45℃. Results：The separation between empaliflozin and its related substances was not less than 1.5. The LOQ was less than 9 ng/mL，and linear range were suitbale for determination. The RSD of repeatability was less than 5.0%. The test solution and reference solution had a good stability in 8 h， the recovery of the impurities was 88.6%~106.1%， and RSD was less than 10%. Conclusion：The method is sensitive and accurate， and is effective for quality control in empagliglozin.