Abstract: OBJECTIVE To develop a method for the determination of related substance in dmbroxol hydrochloride and glucose injection by RP-HPLC. METHODS The analysis was performed on a C18 column. The mobile phase consisted of 0.1 mol·L-1 ammonium acetate solution-methanol (40∶60). The detection wavelength was 246 nm. RESULTS The response factor of impurity B relative to that of ambroxol hydrochloride was 1.47. Ambroxol hydrochloride and related substances could be well separated. The limit of detection of ambroxol hydrochloride and impurity B was 0.75 ng and 1.5 ng, respectively. CONCLUSION This method can be used to simultaneously separate and determine the related substances in ambroxol hydrochloride and glucose injection. It is simple and accurate.