This manuscript describes the determination of oseltamivir (OP) and oseltamivir carboxylate (OC) in rat plasma, cerebrospinal fluid (CSF) and brain and in human plasma and urine using liquid chromatography coupled to tandem mass spectrometry. Threefold deuterated OP and OC served as internal standards. Protein precipitation with perchloric acid was followed by on-line solid-phase extraction and gradient separation on a reversed-phase column. After electrospray ionization, the compounds were detected in positive ion selected reaction monitoring (SRM) mode. Run time was 3.6 min. The lower limits of quantification (LLOQ) were 0.1 ng/mL in rat plasma and CSF, 0.5 ng/g in brain and 1 ng/mL in human plasma and urine. Inter-day and intra-day precisions and inaccuracies in rat matrices were below 10.2% and 13.9% (below 19.0% at LLOQ), respectively. Intra-assay precisions and inaccuracies in human matrices were below 11.7% and 8.9%, respectively. The recoveries were close to 100%, and no significant matrix effect was observed. The method was successfully applied to rat study samples.