Metal-affinity chromatography with Cu²⁺ containing sorbent was used for separation of globin peptides alkylated by sulfur mustard. It was shown that matrix-assisted laser desorption/ionization time-offlight mass spectrometry allowed isolating peptides alkylated by sulfur mustard (HD) at cysteine-126, -94 and glutamic acid-27 with MH⁺ of 1444.62, 1561.66, 1676.78 Da, respectively, from rat globin tryptic digest incubated with 60 μM of HD. An alkylated peptide with MH⁺ of 1444.63 Da was isolated from globin hydrolyzate incubated with 3 μM of HD.