OBJECTIVE： To establish a method for the determination of related substances in bisacodyl raw material and enteric-coated tablet. METHODS：HPLC was performed on the column of Hibar C18 with mobile phase of acetonitrile -20 mmol/L ammonium acetate （acetic acid adjust pH to 5.0）（55 ∶ 45，V/V）， detection wavelength was 265 nm， flow rate was 1.0 ml/min， column temperature was 30℃， and the injection volume was 20 μl. RESULTS： The linear range of bisacodyl was 0.25-5.0 mg/ml（r＝0.999 9）； the limits of detection and quantification were 19-25 ng and 61-68 ng for bisacodyl and impurity A， B， C， D and E； RSDs of precision， stability and reproducibility tests were lower than 2％； recovery was 99.50％-101.00％ （RSD＝0.5％，n＝9）. CONCLUSIONS： The method is specific， sensitive and reproducible， and can be used for the determination of related substance in bisacodyl raw material and enteric-coated tablet.