Abstract: A method for the determination of an important biomarker, alkylated N-terminal valine adduct in hemoglobin, was developed for the retroactive detection of mustard gas exposure. After isolating from hemoglobin, the alkylated globin was treated with a modified Edman degradation method. After extracted with toluene, the biomarker was identified with liquid chromatography-high resolution mass spectrometry (LC-HRMS) and tandem mass spectrometry (MS/MS). The fragmentation pathways were concluded based on the high-resolution of mass spectrometry and second fractured informations. It was found that the protonated precursor ion M+H⁺ fragmented mainly through C—N bond of 2-hydroxyethylthioethyl (HETE) group and valine in the pentafluorophenyl isothiocyanate （PFPITC） derivative. The diagnostic ion m/z 105.036 3 is identified as C₄H₉OS, which is the group of 2-hydroxyethylthioethyl. The PFPITC derivative of the alkylated N-terminal valine is also successfully identified by GC/MS after derivatization with heptafluorobutyrylimidazole(HFBI).