A reversed-phase high performance liquid chromatography coupled with UV detection(RP-HPLC/UV) was developed and validated for the simultaneous determination of four isoflavonoids，including puerarin(PU),3-methoxy puerarin(MP),mirificin(MI) and daidzin(DA),in the bioactive fraction of Naodesheng prescription,ie.a widely used herbal medicine(HM) in China mainly for treatment of cerebral arteriosclerosis,ischemic stroke,sequelae of cerebral hemorrhage and etc.The separation of four compounds was accomplished on a Hyspersil ODS-2 column(250 mm×4.6 mm,5 μm i.d.) by using methanol-water(25∶75) as mobile phase with isocratic elution at a flow rate of 0.9 mL/min.The UV detection wavelength was set at 250 nm,and the column temperature was 25 ℃.The high resolution and reproducible peaks were achieved for four compounds in 20 min.All the calibration curves showed good linearities(r≥0.999 7) over the test concentration range.The relative standard deviations(RSDs) of the method were less than 2.2% for precision,stability and reproducibility assays,and the spiked recoveries were in the range of 96%-102%.The total contents of the four isoflavonoids were determined to be in the range of 37.3%-37.8%in the bioactive fraction of Naodesheng prescription.