A rapid, sensitive, and specific analytical method based on high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for the determination of thalidomide in human plasma. The analyte and internal standard were extracted by liquid-liquid extraction with ether-dichloromethane (3:2, v/v) and separated on a TC-C18 column using methanol-10mM ammonium acetate-formic acid (60:40:0.04，v/v/v) as the mobile phase at a flow rate of 0.9 mL·min-1. The detection was performed using an API 4000 triple quadrupole mass spectrometer under positive ESI mode and completed within 3.0 min. The multiple reaction monitoring (MRM) transitions were m/z 259.1→84.0 for analyte and m/z 195.9 →138.9 for temozolomide. The calibration curve exhibited a linear dynamic range of 2-1500 ng·mL-1 (r > 0.999 1). Intra- and inter-day precisions (as RSD) were 6.8-13.5％ and 4.3-5.0％ respectively, and accuracy (as RE) was 2.0-3.5％. The recoveries and matrix effects were satisfactory in all the biological matrices examined. This method was successfully used in a pharmacokinetic study of thalidomide in Chinese healthy male volunteers receiving an oral administration of 200mg dose.