A fully automated high-throughput liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed for the determination of roxithromycin in human plasma. The plasma samples were treated by liquid-liquid extraction (LLE) in 2.2 mL 96-deep-well plates. Roxithromycin and the internal standard clarithromycin were extracted from 100 microL of human plasma by LLE, using methyl t-butyl ether as the organic solvent. All liquid transfer steps were performed automatically using robotic liquid handling workstations. After vortexing, centrifugation and freezing, the supernatant organic solvent was evaporated and reconstituted. Sample analysis was performed by reversed-phase LC-MS/MS, with positive ion electrospray ionization, using multiple-reaction monitoring. The method had a very short chromatographic run time of 1.6 min. The calibration curve was linear for the range of concentrations 50.0-20.0x10(3) ng mL(-1). The proposed method was fully validated and it was proven to be selective, accurate, precise, reproducible and suitable for the determination of roxithromycin in human plasma. Therefore, it was applied to the rapid and reliable determination of roxithromycin in a bioequivalence study after per os administration of 300 mg tablet formulations of roxithromycin.