Objective Hair shaft is one kind of common biological evidences at the crime scene. However, it fails to play an important role in the crime investigation due to lack of effective method of individual identification. The single amino acid polymorphisms (SAPs) in the hair shaft proteome contain information of individual genetic differences.Methods In order to study SAPs in the hair shaft, the proteome of single 2 cm hair shaft samples (6 people, 2 hairs per person) were extracted using ionic liquid following with LC-MS/MS detecting. The protein composition of the hair shaft was analyzed. A custom SAP protein sequence database was built for East Asian population as the searching database. Based on the custom SAP and SNP corresponding annotation table information, the nsSNP profiles were imputed corresponding to SAP. The accuracy of SAP was verified by comparing the imputed nsSNP profiles from SAP with nsSNP profiles obtained from the whole exome sequencing. The validated SAPs were used to calculate the random matching probability.Results In 12 samples, 321 SAPs were obtained, with an average of (131±17) for each sample. The value of random matching probability for 6 people ranged from 1.4×10^-4 to 1.0×10^-9.Conclusion In this paper, a method for detecting SAP in hair shaft proteins of East Asian populations was established, and the ability of individual identification application was verified, which provided a powerful tool and new ideas for individual identification of hair shaft in forensic science.