OBJECTIVE: To develop a fast and sensitive high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) for determining thymosin β4 in rat plasma. STUDY DESIGN: The internal standard analyte was extracted from the rat plasma by cold methanol precipitation. Then 10 μL of sample supernatant was utilized for detection by UPLC-MS/MS method. The reliability of the method was evaluated by the selectivity, calibration curve, lower limit of quantification, precision and accuracy, matrix effect, recovery, and carry-over. RESULTS: Within the concentration range of 0.1–100 ng/mL, the linear correlation coefficients obtained from the calibration curves were all over 0.997. CONCLUSION: The method has good retention time and peak area repeatability, and the area ratio of residual area to lower limit of quantification is 5.4%, which meets the requirements of the guidelines for nonclinical pharmacokinetic studies of drugs. © Science Printers and Publishers, Inc.