Abstract: OBJECTIVE To establish an HPLC method for the determination of acetaminophen in the plasma of volunteers who were administrated the compound tablets of acetaminophen, pseudoephedrine hydrochloride and dextromethorphan hydrobromide or acetaminophen, pseudoephedrine hydrochloride, diphenhydramine hydrochloride and dextromethorphan hydrobromide.METHODS Proteins in plasma samples were precipitated by 6% perchloric acid. A Zorbax Eclipse XDB-C18 was used with the mobile phase of 0.02 mol·L-1 ammonium formate solution(contained 0.2% formic acid)-methanol-acetonitrile (91∶4.5∶4.5) at a flow rate of 1.0 mL·min-1. The detection wavelength was set at 245 nm. RESULTS The endogenous substance in plasma did not interfere with the determination of the samples. The linear range was 0.1～20 μg·mL-1(r=0.999 6), and the limit of quantitation was 0.1 μg·mL-1. The method recovery was 99.8%-101.5%. Both within-day RSD and between-day RSD were less than 12%.CONCLUSION The method was simple and accurate. It was suitable for the pharmacokinetic study of acetaminophen.