【Abstract】 Objective To optimize the enzymatic digestion of proinsulin aspart and establish a purification protocol to obtain insulin aspart with high purity and bioactivity. Methods The enzymes are trypsin and carboxypeptidase B, and the substrate is proinsulin aspart which is expressed in E.coli with an extra peptide C to assist protein’s refolding. The reaction is operated with the mass ratio of 1: 1000 (enzyme: protein), at room temperature for 230 min. Ion exchange chromatography of SP FF was applied to purify the digested products with a gradient elution of 0100% in 15 min. Results RPHPLC and SDSPAGE analysis showed that the digestion would complete in 30 min. SP FF can separate insulin aspart from peptide C. The molecular weight of the purified insulin aspart is same as the theoretic value. The purity of insulin aspart was greater than 95% and the yield was 30%. Conclusion The enzymatic digestion and purification methods could be adopted for insulin aspart’s large production.