A novel strategy to annotate nsSNP-peptides in human liver proteome based on LC-ESI-MS/MS and peptide database search was proposed. Totally 115 nsSNP-peptides in human liver proteins were annotated using our method. Among them, 42 peptides were found to be amino acid mutation, 73 peptides were wild type, 5 peptides were interpreted with both mutation and wild type. The function of nsSNP-peptide was predicted using SIFT algorithm, and 2 nsSNPs were predicted to be damaged for protein function. The results here show that the strategy is very effective for annotation of nsSNP at peptide level.