This paper aims to establish an accurate and sensitive LC-MS method for determination of tyroserleutide in rats' blood, and to investigate the pharmacokinetics of tyroserleutide after i.v. injection in rats. The rats were anesthetized and the cannula was inserted into the carotid artery. Tyroserleutide was administrated to rats by Tyr-ser-Len i.v. injection with the dose of 200 μg/kg, 400 μg/kg, and 800 μg/kg, then the blood samples were collected. After disposals of the samples, the concentration of tyroserleutide was determined by LC-MS method with pos. ion SIM detection, using YSW as internal standard, and the CHCH3CN-H2O-H3COOH as elution system. The pharmacokinetics parameters were calculated The tyroserleutide was rapidly degraded, with a short half life (about 20 s). The LC-MS method has proved to be accurate and sensitive, and the results are reliable. The tyroserleutide has a short half life after bolus i.v. injection. The dosage regimen is not suggested in clin. trial and another new regimen needs to be further investigated.