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.