Abstract: OBJECTIVE To establish a method for determination of three main conjugated estrogens in pregnant mare urine of Xinjiang by substitute reference substance by HPLC. METHODS Sodium estrone sulfate was selected as the substitute for reference substance of sodium equilin sulfate and sodium 17α-dihydroequilin sulfate. The relative correction factors(RCF) of sodium equilin sulfate and sodium 17α-dihydroequilin were measured by HPLC under different conditions. Assay of three main conjugated estrogens in pregnant mare urine of Xinjiang was determined by substitute reference and the relative correction factors. RESULTS The quantitative results of the new method were almost consistent with the results of conventional HPLC. CONCLUSION The substitute reference substance is adopted in HPLC determination of three main conjugated estrogens in pregnant mare urine of Xinjiang. The new method is economical and practical.