A semi-automated liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the determination of glimepiride in human plasma. The plasma samples were treated by liquid-liquid extraction (LLE) in 1.2 mL 96-well format micro-tubes. Glimepiride and the internal standard (IS) glibenclamide were extracted from human plasma by LLE, using a mixture of ethyl acetate/diethyl ether 5050 (v/v) as the organic solvent. After vortexing, centrifugation and freezing, the supernatant organic solvent was evaporated. The analyte and IS were dissolved in a small volume of a reconstitution solution, an aliquot of which was analyzed by reversed-phase LC/MS/MS with positive ion electrospray ionization, using multiple reaction monitoring. The method proved to be sensitive and specific for both drugs, and statistical evaluation revealed excellent linearity for the range of concentrations 2.0-500.0 ng/mL with very good accuracy and inter- and intra-day precisions. The proposed method enabled the rapid and reliable determination of glimepiride in pharmacokinetic or bioequivalence studies after per os administration of a 3 or 4 mg tablet of glimepiride.