An　increasing　number　of　peroxynitrite-mediated　fibrinogen　nitrifications　have　been　associated　with　thrombotic　diseases.　However,　few　reports　related　to　priority　nitrified　fibrinogen　injury　sites　exist.　In　this　paper,　an　improved　method,　which　simulated　the　environment　in　vivo,　was　used　to　inspect　the　structural　changes　of　fibrinogen　treated　with　peroxynitrite　and　LC-MS/MS　in　order　to　investigate　the　fibrinogen　injury　sites.　The　SDS-PAGE　results　indicated　that　gamma　subunits　of　Fg　were　vulnerable　to　oxidative/nitrative　modifications　induced　by　peroxynitrite.　An　in-depth　analysis　of　fibrinogen　gamma　chain　(Fg　I)　nitration　site　identification　and　susceptibility　to　peroxynitrite-utilizing　LC-MS/MS　strategy　was　performed.　Based　on　a　large　dataset,　the　results　indicated　the　priority　injury　sites　during　database　searching　were　Y96,　Y262,　Y274,　Y348,　and　Y363;　these　results　could　be　applied　to　biomedical　studies.