The possibility of interaction of 0.1 mg/mL acetylsalicylic acid (ASA) with purified human and rat globin proteins for 24 h in vitro was investigated. It was shown that following the interaction with ASA rat globin is modified at Lys17, Lys57, Lys91, and Lys140 amino acid residues of the α-subunit as well as at Lys18 and Lys77 of the β-subunit, whereas human globin is acetylated at Lys17, Lys41, Lys57, and Lys91 residues of the α-subunit as well as Lys18, Lys96, and Lys133 of the β-subunit of the protein. Incubation of human whole blood with 0.1 mg/mL ASA for 3 h followed by globin isolation led to the identification of acetylated Lys17 and Lys57 lysine residues of the α-subunit of human globin.