A method for the assay of carbocysteine and its related substances by HPLC was established. A RP-HPLC method was used with C18 column (4.6 mm × 150 mm, 5 μm); the mobile phase was consisted of potassium phosphate monobasic of 0.68% and 1-hexanesulfonic acid sodium salt of 0.05%, pH adjusted to 2.5 with the H3PO4, and the detection wavelength was 215 nm. The linear range of pazufloxacin mesilate was 0.44-1.3 mg/mL-1 (r = 0.9999). The average recovery was 99.5% with the RSD of 1.1% (n = 9). The limit of detection was 8.27 ng. The chromatog. peaks of cidofovir and its related substances were separated completely. The method was convenient, specific, and reproducible for the assay of carbocysteine and related substances.