Although racemic sibutramine has been widely used for the treatment of obesity, its enantioselective detection method has not been elucidated in human plasma. In this report we introduce a validated analytical method for the determination of sibutramine and its two active metabolites, desmethylsibutramines using LC-MS/MS. R- and S-isomers of those compounds in human plasma were extracted using diethyl ether-hexane (4:1, v/v) followed by an addition of NaOH solution. After removing the organic layer, the residue was reconstituted in the mobile phase 10mM ammonium acetate solution adjusted to pH 4.0 with acetic acid-acetonitrile (94:6, v/v). Both isomers in the extract were separated on a Chiralcel AGP chiral stationary-phase column and were quantified in a tandem mass spectrometry. The assay method was in accordance with FDA regulations for the validation of bioanalytical methods. This method was successfully used to profile the plasma concentrations of the stereoisomers of sibutramine and its two active metabolites with time in healthy volunteers.