The radioisotopic tracing method of Hirulog-like peptide（HLP） was established, wihch could be a reference for the pharmacokinetic analysis in small molecule polypeptide research.（）125I-HLP was prepared by Iodogen method.The radiochemical purity and stability in vitro of（）125I-HLP were determined by HPLC and SDS-PAGE.The radiochemical purity of(（）125I-HLP) was shown above 95% and was stable for 53 hours.It is the first time using SPE to separate the labeled compound from radioiodide and small molecule polypeptides.The HLP recovery was（92.60±6.31）% by SPE and the coefficient of variation was lower than 10% in a concentration range from 10 to 100 000 ng/mL.The detectable limitation reached 52.79 ng/mL.
