Abstract : The purpose of this article was to establish methods for content determination of acetaminophen. We used High-performance liquid chromatography and ultraviolet spectrophotometry. Chromatographic conditions were C18 column and methanol-water (volume ratio 70:30) as the mobile phase. The flow rate was 1.0 mL / min and the detection wavelength was 250nm. In the range of 5~30 μg/mL, acetaminophen’s concentration showed a good linear relationship (r = 0.9997). The recovery ratio was 99.6% (RSD = 1.9%). The p-aminophenol was detected under the same conditions and its linear range was 0.1μg/mL~1.2μg/mL. UV spectrophotometry was used to determine the content of acetaminophen. In the range of 1~10ug/mL, the concentration showed a good linear relationship and the average recovery ratio was 99.9%(RSD = 1.1%). The two methods were simple, sensitive, reproducible and accurate, which can be used in the content determination of acetaminophen.