OBJECTIVE To develop a RP-HPLC method for the determination of norcantharidin and its related substances.METHODS A chromatography was carried out on a YWG-C₁₈ column(4.6 mm×250 mm,5 μm),using a mobile phase consisting of methanol-0.016 mol·L^-1 potassium dihydrogen phosphate(30∶70)(adjusted to pH 3.1 with phosphoric acid).The detection was carried out with a UV detector at 213 nm.The flow rate was 0.9 mL·min^-1.RESULTS Norcantharidin,maelic anhydride,dehydronorcantharidin and degradation products were separated.The calibration curves of norcantharidin,maelic anhydride and dehydronorcantharidin were linear(r=0.999 8,0.999 9,0.999 7).The detection limits were 10.0 ng for norcantharidin,0.08 ng for maelic anhydride and 0.63 ng for dehydronorcantharidin,respectively(S/N=3).The recoveries of norcantharidin,maelic anhydride and dehydronorcantharidin were 99.94%,100.43% and 100.03%,respectively(n=9).The norcantharidin curves were linear in the range of 0.126～4.044 g·L^-1.CONCLUSION This method is convenient,accurate and suitable for the determination of norcantharidin and related substances.