In the present study, an automated, 96-well format LC-MS/MS method for the determination of anastrozole in human plasma was developed and fully validated. Within method development procedure, atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) were compared in terms of sensitivity and specificity, with the former proven to be more appropriate and thus being chosen for analyte ionization. In addition, the effect of declustering potential (DP) and collision energy (CE) in sensitivity was, as well, studied and compared between APCI and ESI source employment. Samples were treated with an acetonitrile (ACN) protein precipitation step followed by liquid-liquid extraction (LLE) with methyl t-butyl ether (MTBE) as the organic solvent, using omeprazole as the internal standard (IS). The statistical evaluation for the APCI protocol revealed excellent linearity, accuracy and precision values for the range of concentrations 0.100-100 ng/mL. The method proposed involves the lowest plasma volume so far reported (190 microL), as well as the shortest run time (1.6 min) and along with the employment of two robotic liquid handling systems enabled the rapid and reliable determination of anastrozole in a bioequivalence study (>1000 plasma samples) after per os administration of 1mg tablet within a 4-day period of time.