Hirudin, a potent clinical thrombin inhibitor from Hirudo medicinalis, consists of 65 amino acids in a single chain. In this paper, we systematically synthesize a series of C-terminal (desulfo hirudin45-65) peptides substituted by 20 natural L-amino acids via the Multipin method. The resulting peptide library is subsequently screened using an alpha-thrombin-mediated fibrinogen clotting assay and alpha-thrombin-induced amidolytic hydrolysis assay.