A specific and sensitive liquid chromatography–tandem mass spectrometry (LC/MS/MS) in the negative electrospray ionization mode was developed and validated in order to analyze 3α-hydroxytibolone (3α-OH-tibolone), major metabolite of tibolone in human plasma sample using only 200 μL of plasma. 3α-OH-tibolone and 3α-OH-tibolone-d6 (internal standard, IS) in plasma were derivatized with p-toluenesulfonyl isocyanate after extraction using ethyl acetate. The dye residue and by-products was excluded using HLB Oasis SPE cartridge after derivertization. Separation of derivatized 3α-OH-tibolone and IS was performed on a C18 column within 6 min. The assay showed linearity in concentration ranges of 0.2–20 ng/mL. Intra-day precision and accuracy ranged from 0.77 to 5.0 % and from 98.0 to 100.56 %, respectively. Inter-assay precision and accuracy ranged from 1.49 to 5.00 % and from 99.75 to 100.94 %, respectively. A specific, reproducible and sensitive LC/MS/MS method for quantification of 3α-OH-tibolone in human plasma was successfully applied to evaluate the bioequivalence of tibolone in human volunteer following single oral administration of 5 mg tibolone.