Abstract: OBJECTIVE To establish a method to determine the content and related substances of anisodamine hydrobromide injection. METHODS The analysis was performed on an Intersil ODS-3 C₁₈ column(250 mm×4.6 mm, 5 μm) and the mobile phase consisted of 0.01 mol·L^-1 potassium dihydrogen phosphate solution(with 0.15% triethylamine, adjust pH to 6.5 with phosphoric acid) and methanol(70∶30). The flow rate was 1.0 mL·min^-1. The column temperature kept at 35 ℃, and the detection wavelength was 220 nm. RESULTS Main peak, atropic acid, atropine and other individual impurity peaks were separated. The anisodamine hydrobromide was linear in the range of 0.25-2.5 mg·mL^-1(r=1.000), atropine and tropic acid were linear in the range of 1-100 μg·mL^-1(r=0.999). The average recoveries of anisodamine hydrobromide, atropine and tropic acid were 99.5%-100.8% during tests in their high, middle and low concentrations. The anisodamine hydrobromide’s detection limit was 0.2 ng and the limit of quantification was 0.7 ng. The atropine and tropic acid’s detection limit were 0.15 ng and the limit of quantification was 0.17 ng. CONCLUSION The method is rapid, accurate, sensitive and reliable for content determination of anisodamine hydrobromide injection and its related substances.