OBJECTIVE： To establish a method for content determination of thymosin α1 in Thymopolypeptides enteric-coated tablets. METHODS： HPLC-MS/MS method was adopted. The determination was performed on Luna C18（2） with mobile phase consisted of 0.1％ formic acid-acetonitrile （gradient elution） at the flow rate of 0.7 mL/min. The column temperature was set at 30 ℃， and sample size was 20 μL. ESI was used with ion spray voltage of 4.5 kV， sheath gas flow rate of 60 arb， aux gas flow rate of 30 arb， sweep gas flow rate of 10 arb and capillary temperature at 320 ℃.The working mode was positive ion monitoring mode. RESULTS： The linear range for thymosin α1 was 1-1 000 ng/mL （r＝0.999 9）. The limit of quantitation was 1 ng/mL. The limit of detection was 0.1 ng/mL. RSDs of precision， stability and reproducibility tests were all lower than 2.0％. The recoveries were 95.0％-98.0％ （RSD＝1.2％，n＝6）， respectively. CONCLUSIONS： The method is simple， rapid， sensitive and accurate， and can be suitable for simultaneous determination of thymosin α1 in Thymopolypeptides enteric-coated tablets.