Abstract: An accurate RP-HPLC method for the determination of 10-hydroxy-3,9-dimethoxypeterocarpan, (3R)8,2'-dihydroxy-7,4'-dimethoxyisoflavan, formononetin (7-hydroxy-4'-methoxyisoflavone), 8,3'-dihydroxy-7,4'-dimethoxy isoflavone,2' -hydroxy-3',4'-dimethoxyisoflavan-7-O-glucopyranoside and alycosin (7,3'-dihydroxy-4'-methoxyisoflavone) in the hairy root cultures of Astragalus membranaceus was developed. Among them, 10-hydroxy-dimethoxypeterocarpan, (3R)8,2'-dihydroxy-7,4'-dimethoxyisoflavan and 8,3'-dihydroxy-7,4'-dimethoxyisoflavone were new compounds in Astragalus membranaceus. The analytical column was Nucleosil C₁₈. The mobile phases were methanol—water=3∶2 and 1∶1 (v/v). The detection wavelengths were 254 nm and 280 nm. Calibration graphs were rectilinear between 2.5 μg and 12.5 μg. The recoveries were between 96.47% and 103.33%. The relative standard deviations (RSDs) of mesurement precision test were 2.57%～6.52%. The flavonoid contents in the hairy root cultures were between 0.0005% and 0.0065% (dry weight).