[Objective] Hyperoside in Acanthopanax setchuenensis Harms ex Diels and A. leucorrhigus var. fulvescens Harms & Rehd was identified qualitatively by LC-MS,and a RP-HPLC method was established to determine the content of hyperoside in their different medicinal parts. [Methods] The analysis was carried out at 30 ℃ on a Kromasil C18 column eluted with a mobile phase consisted of methanol(A) and 0.5% phosphate in water(B). The detection wavelength was 358 nm,and the flow rate was 1.0 mL/min. [Results] The calibration curve was linear in the concentration range of 2.50-25.00 μg / mL,the regression equation was Y = 4 × 10~6 X + 1. 327 × 10~4( r = 0. 999 9). The average recovery was 100. 25%,with RSD of 0. 96%( n = 9). [Conclusions] This method was simple,accurate and replicable,and it was useful for the study of the quality control of A. setchuenensis Harms ex Diels and A. leucorrhigus var. fulvescens Harms & Rehd.