In order to develop a method for the determination of the content of acetamide in hydrochloride. The High-performance liquid chromatography (HPLC) was used with an Agilent ZORBAX SB-C18 column (25 cm×4.6 mm, 5 μm) as the analytical column; column temperature: 30 ℃; detection wavelength: 230 nm; flow rate: 1.0 mL/min; injection volume: 20 μL; mobile phase: buffer solution (0.02 mol/L sodium dihydrogen phosphate solution with 0.1% triethanolamine, diluted with dilute phosphoric acid to pH=3.0): acetonitrile = 65∶35; diluent: mobile phase. Results show that acotiamide hydrochloride had a retention time of 5.45 min, ran smoothly, and there was no interference from impurities. Acotiamide Hydrochloride had a good linear relationship with a linear equation of y=58.947x-0.201 7 (r2=0.9997) at a concentration of 0.20 ~ 0.60 mg/mL. The detection limit was 0.121 μg/mL, the quantitative limit was 0.452 μg/mL; the stability test of the sample solution showed that the stability was good within 24 h, with an RSD of 0.23 (n=7); the RSDs of repeatability and intermediate precision were 0.10% (n=6) and 0.14 (n=12), respectively, indicating good repeatability and intermediate precision; the RSD of the durability test was 1.05% (n=9), and the RSD of the content determination was 0.13% (n=9); the content determination results of three self-prepared samples were 99.11%, 99.16%, and 99.09%.