In this paper, p-toluenesulfonyl isocyanate has been reported as a novel derivatization reagent with strong nuclephilic reactivity for the hydroxyl compounds. The derivatization for the two pharmacologically active 3-hydroxyl metabolites, 3alpha-hydroxyl-7-methyl-norethynodrel and 3beta-hydroxyl-7-methyl-norethynodrel by p-toluenesulfonyl isocyanate can be accomplished in 2 min under room temperature. The offline derivatization procedure introduced an easily ionizable sulfonylcarbamic ester moiety to the metabolites. This greatly improved the analyte's sensitivity in negative electrospray ionization and enabled us to achieve the desired lower limit of quantitation at 100 pg/ml in plasma. Therefore, a sensitive high performance liquid chromatography-mass spectrometry (HPLC-MS) method for the analysis of the two stereo isomers was developed. The method had been validated to be accurate, precise, and sensitive, and can be used for the metabolism pharmacokinetic study of tibolone in human subjects.