Liquid chromatography combined with electrospray ionization tandem mass spectrometry positive ion mode is used for the determination of glimepiride in human plasma. Gliclazide was used as the internal standard. The chromatographic separation was performed on a ACE 5 Cl 8, 50 x 4 mm, 5 pm column at 30 degrees C with mobil phase consisting of 200 mL water, 450 mL acetonitrile, 350 mL methanol, 0.6 mL glacial acetic acid with a 0.5 mL min(-1), flow rate. The work-up procedure involved a liquid-liquid extraction of the compounds. Mass spectrometric data were acquired in single ion monitoring of the ions 324.11 > 127.25 and 491.16 > 352.08 for glimepiricle and gliclazide, respectively. The method was validated in the concentration range of 5-1,000 ng mL(-1). Retention times of glimepiricle and gliclazide were 1.65 and 1.36 min, respectively. The run time was 2.5 min. The method was found suitable to analyse human plasma samples for application in pharmacokinetic, pharmacodynamic, bioavailability/bioequivalance studies.