Abstract: This paper describes the use of high performance liquid chromatography (HPLC) to determine the hyperin in the leaves of Acanthopanax senticosus on a C18 chromatogram column. Methanol:0.025 mol.L-1 phosphoric acid solution (V/V, 55:45) was used as the mobile phase (the mixture was adjusted with triethylamine to pH 3.0-3.2), and the detection wavelength was 360 nm. The sample was extracted with methanol by the ultrasonic extraction method. Hyperin content showed a symmetrical peak shape and reached the baseline separation with the peak content of other chemicals. The average recovery was 104% and the relative standard deviation (RSD) 5.38%. This study provided evidence of an accurate and credible method of determining the content of hyperin in the leaves of A. senticosus.