Abstract: OBJECTIVE To establish a method for determination the content of tiamulin fumarate. METHODS RP-HPLC was adopted, the column was Agilent C18 (150 mm×4.6 mm, 5 μm), the mobile phase was 0.1 mol·mL-1 ammonium carbonate buffer (add 10 g ammonium carbonate to a 1 000 mL volumetric flask and dissolve in about 800 mL of water. Add 24 mL 6% of perchloric acid and dilute with water to volume) -acetonitril-methanol (27∶23∶50). UV dector wavelength was 212 nm. RESULTS The calibration is linear between 0.25-0.75 mg·mL-1. The mean recovery was 99.3%, RSD%=1.2%. CONCLUSION This method inspecting tiamulin fumarate is rapid, accurate and reproducible.