A fully automated high-throughput liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous quantification of simvastatin (SV) and simvastatin acid (SVA) in human plasma. Plasma samples were treated by acetonitrile (ACN) addition for protein precipitation (PP) and subsequent two-step liquid-liquid extraction (LLE) in 96-deepwell plates, using methyl t-butyl ether (MTBE) as the organic solvent. ACN addition step was proven to enhance method sensitivity, as well as producing cleaner samples for injection. Lovastatin (LV) and lovastatin acid (LVA) were used as internal standards (IS) for SV and SVA quantification respectively. A relatively small plasma volume (300 microL) was employed and all procedure liquid transfer steps were performed automatically, by the use of robotic liquid handling workstations. Both electrospray (ESI) and atmospheric pressure chemical ionization (APCI) sources were applied and compared for LC-MS/MS sample analysis, with ESI proven to be more sensitive for the specific analytes. Polarity switch (from negative to positive ionization mode) was performed during the same analytical run, so as for the simultaneous SV and SVA determination to be possible. The method had a short sample preparation time, as well as a chromatographic run time of just 1.9 min, the shortest so far reported for SV determination. It was validated and fulfilled all preset criteria for sensitivity, specificity, linearity (0.100-40.0 ng/mL), inter- and intra-accuracy and precision for both molecules. The proposed method was applied to the rapid and reliable simultaneous determination of SV and SVA in a bioequivalence study, after per os administration of a SV tablet (80 mg).