Aim: To establish an HPLC method for the determination of secnidazole and its related substances, and to elucidate the structure of the main related substances. Methods: HPLC was performed on an Agilent C_(18) (150 mm × 4. 6 mm, 5 μm) column. The mobile phase consisted of acetonitrile and 0. 1% formic acid solution( 10 : 90). The detection wavelength was set at 320 nm and the column oven was maintained at 30 ℃. The mass spec-trometry detector was equipped with an ESI source with a temperature of 350 ℃, and set at positive ion monito-ring model The spray chamber pressure was 0. 34 MPa. The drying gas flow rate was 10 L/min. Results: Sec-nidazole was completely separated from the impurities. The calibration curve of secnidazole was linear over the range of 0. 10-1 500. 0 μg/mL with the correlation coefficient of 0. 999 9. The contents of secnidazole in the sam-ples were 100.5%, 100.3% and 100.7%, respectively, and those of the related substances were 0.463%, 0. 488% and 0.465%, respectively. The three related substances were identified by HPLC-UV-MS~n as 2-methyl-5-nitroimida-zole and the isomerides of secnidazole, respectively. Conclusion: The developed method is reliable for the quality control and determination of related substances of secnidazole.