Nucleoprotein (NP), the structural component of ribonucleoprotein complex of avian influenza virus, performs multiple essential functions in the regulation of viral RNA synthesis and in the control of nuclear traffic of viral proteins. Mutations have often been found in NP, some of which are relevant to viral survival strategies. In this study, we used nanospray-MS/MS to analyze tryptic digestion of nucleoprotein of avian influenza virus (H5N1) and to identify three mutated peptides. The MS/MS analyses allowed the confident determination of the three mutated amino acid residues F313Y, I194V and V408I/L in the mutated peptides of LLQNSQVYSLIRPNENPAHK, GVGTMVMELVR and ASAGQI/LSVQPTFSVQR, respectively.