The disturbance of estradiol level might induce the occurence of some diseases, including cancer. Estradiol is mainly metabolized through the conjugation reactions, including the sulfation and glucuronidation reactions. The present study tried to evaluate the inhibition of estradiol glucuronidation by the major ingredients of Tripterygium wilfordii Hook F. demethylzeylasteral. Selective ion monitoring at negative ion mode ([M⁺ H⁻] = 447) was employed to monitor the two glucuronides of estradiol. The reaction rate was determined through comparison of peak area of these two glucuronides. Lineweaver-Burk plot and Dixon plot were utilized to determine the inhibition kinetic type, and the inhibition kinetic parameters (K i) were calculated using the second plot. Competitive inhibition of demethylzeylasteral towards the formation of two glucuronides of estradiol was demonstrated, and the K i values were calculated to be 453.3 and 110.9 μM, respectively. All these results will remind us the risk of elevated serum concentrations of estradiol due to the inhibition of estradiol glucuronidation by demethylzeylasteral.