OBJECTIVE： To establish a method for the determination of related substances in tosufloxacin cyclizaton. METHODS： HPLC was performed on the column of YMC-Pack ODS A-312 with mobile phase of acetonitrile-water（5 ∶ 3，V/V） at a flow rate 1.2 ml/min， detection wavelength was 250 nm， column temperature was 25℃ and injection volume was 10 μl. RESULTS： The main component and related substances could be baseline separated by the conditions； the linear range was 0.04-1.8 μg/ml （r＝0.999 9）；the limit of quantification was 40.56 ng/ml and the limit of detection was 6.3 ng/ml； RSDs of precision， stability and reproducibility tests were lower than 3％； recovery was 99.88％-100.68％（RSD＝0.29％，n＝9）； the result of total impurities of 6 batches of sample were less than 0.1％. CONCLUSIONS： The method is specific， sensitivity with high accuracy and precision， and can be used for the determination of related substances in tosufloxacin cyclizaton.