A high performance liquid chromatographic method was established to simultaneously quantify the lenalidomide, vorinostat and dexamethasone in rat plasma. The method was successfully applied to the plasma pharmacokinetics of lenalidomide,vorinostat and dexamethasone after oral administration to rats. The LC column was Agilent Zorbax Eclipse Plus C₁₈(100 mm×2.1 mm,1.8 μm). The mobile phase comprised of acetonitrile and water containing 0.1% formic acid+5mM ammonium acetate. Flow rate was 0.6 mL/min. Column temperature was 35 oC. Sample injection volume was 2 μL. The quantification was obtained by scanning with multiple reaction monitoring (MRM) via the positive electrospray ionization (ESI^₊) source.The correlation coefficients of the nobiletin and tangeretin, respectively were over 0.995. The RSD values of the inter- and intra- precisions were less than 7.4%. The accuracy ranged from 94.7% to 106.7%. The matrix effect values were sufficiently minimized to 92.3%~109.7%.The average recoveries from rat plasma were 87.4% to 103.6%. The validated method was further applied to the determination of lenalidomide, vorinostat and dexamethasone in actual rat plasma. The method is rapid and will provide helpful information for the development of lenalidomide, vorinostat and dexamethasone.