OBJECTIVE： To establish a method for determination of the related substances in Sertaconazole nitrate vaginal tablet. METHODS: HPLC was performed on the column of Hypersil BDS C18（150 mm×4.6 mm， 5 μm） with mobile phase of 0.5％ Ammomium acetate solution-acetonitrile-methanol（15 ∶ 42.5 ∶ 42.5，V/V/V） at a flow rate of 1.0 ml/min， detection wavelength was 220 nm， column temperature was 25 ℃， and the injection volume was 20 μl. RESULTS: The related substances in Sertaconazole nitrate vaginal tablet can be well separated； the liner range of sertaconazole nitrate was 12.24-28.56 μg/ml （r＝0.999 9）； RSDs of precision， stability and reproducibility tests were lower than 1％； recovery was 99.38％-99.80％ （RSD＝0.14％，n＝9）. CONCLUSIONS： The method is accurate and reliable with high sensitivity and strong specificity， and can be used for the related substances in Sertaconazole nitrate vaginal tablet.