A rapid, sensitive and specific liquid chromatography-electrospray-tandem mass spectrometric (LC-ESI-MS/MS) method for the simultaneous detection and quantitation of methylprednisolone acetate (MPA) and methylprednisolone (MP) in rat plasma, using a triple-stage quadrupole, has been developed and validated. MP-D(2) was used as internal standard (IS) and acetonitrile was added to plasma samples for protein precipitation. After extraction with dichloromethane, the analytes were separated on a C-12 reversed-phase column by isocratic elution (6min at a flow rate 0.2mLmin(-1)) with water containing 0.01% formic acid (A) and acetonitrile (B) (50:50, v/v). Quantitation was performed in positive ion multiple reaction monitoring (MRM) mode by applying the following precursor-to-product ion transitions: MPA m/z 417-->135+161+253; MP m/z 375-->135+161+253; IS m/z 377-->135+161+253. The method, validated over the concentration range 6-600ngmL(-1), has been shown to meet the current requirements of bioananalytical validation, providing satisfactory results in terms of linearity, recovery, intra-day and inter-day precision and accuracy. The lower limit of quantitation (LLOQ) was 6ngmL(-1) for both the analytes (0.080 and 0.072pmol injected for MP and MPA, respectively). The method was successfully applied to monitor the plasma levels of MPA and MP following intra-articular (IA) injections of a low MPA (Depo-Medrol((R))) dose in rats.