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Objective:To establish an RP-HPLC method for determination of diosgenin in Rhizoma Dioscoreae Nipponicae,giving the reference for controlling the intrinsic quality.Methods:Firsdy,Diosgenin was extracted by two-phase acid hydrolysis from dry herbal material.Secondly,sample solutions were analyzed on Agilent Zorbax Extend C18 column (4.6 min×250 mm,5 μm)at 40℃.The diosgenin was separated with acetonitrile-pure water (94∶6) as a mobile phase at a flow rote of 1.0 mL·min-1 under a ultraviolet detection wavelength of 203 nm.Results:Diosgenin could be separated obviously from the interferential components in sample solutions.The linearity of the calibration cure (r=0.9999) was good within the range of 1.055-21.10 μg.RSD of repeated experiments (n=6) was 1.7% with average recovery (n=6) reached 96.8% (RSD=1.5%).The average content (n=6) of diosgenin in Rhizoma Dioscoreae Nipponicae was 1.61% (RSD=1.7%).Conclusions:This method can sensitively make a quantitation of diosgenin with a simple pretreatment,and has a broad linear range and good repeatability,which makes it suitable for determination of diosgenin in Rhizoma Dioscoreae Nipponicae.