Abstract: The degummed silk was hydrolyzed using sulfuric acid and then after a series of decoloration, filtration, sedimentation, etc.to prepare fibroin peptide. Hydrolysis process conditions were optimized by using orthogonal test L 9 ( 34) . Then the molecular weight distribution of the silk protein hydrolyzate was determined by high-performance liquid chromatography. The molecular weight distribution curve was obtained as the relationship between molecular weight of standard and elution time.The results showed that silk fibroin peptide yield was the highest when silk protein was hydrolyzed under the conditions of solid-liquid ratio of 1∶40, the sulfuric acid concentration of 40%, hydrolysis temperature of 80℃, hydrolysis time 8h and the molecular weight distribution less than 3500u.The repetition of result was good. In conclusion, it suggested that it was an efficient way to analysis distribution of polypeptide compounds.It provided a new way in the study of silk peptide.