Objective:A high performance liquid chromatography mass spectrometry (LC-MS) 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. Methods:The LC column was Agilent Zorbax Eclipse Plus C18(100 mm ×2.1 mm,1.8 μm). The mobile phase was comprised of acetonitrile and water containing 0.1% formic acid+5 mmol/L ammonium acetate. The gradient profile was used. Flow rate was 0.6 mL/min. Column temperature was 35 ℃. Sample injection volume was 5 μL. The quantification was obtained by scanning with multiple reaction monitoring(MRM)via the positive electrospray ionization(ESI + ) source. Results:The correlation coefficients of the lenalidomide,vorinostat and dexamethasone,respectively were over 0.995. The relative standard deviation(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%. Conclusion:The validated method is further applied to the determination of lenalidomide,vorinostat and dexamethasone in plasma. The method is rapid and economical,and can provide helpful information for pharmaceutical analysis.