Malondialdehyde (MDA) is the most abundant α,β-unsaturated aldehyde generated from endogenous peroxidation of polyunsaturated fatty acids and is present in cigarette smoke. Post-translational modifications of blood hemoglobin can serve as biomarkers for exposure to chemicals. In this study, two types of MDA-induced modifications, the N-propenal and the dihydropyridine (DHP), were identified at multiple sites in human hemoglobin digest by the highresolution mass spectrometry. The N-propenal and the DHP types of modification led to the increase of 54.0106 and 134.0368 amu, respectively, at the N-terminal and lysine residues. Among the 21 MDA-modified peptides, 14 with dose−response to MDA concentrations were simultaneously quantified in study subjects by the nanoflow liquid chromatography nanoelectrospray ionization tandem mass spectrometry under selected reaction monitoring (nanoLC−NSI-MS/MS-SRM) without prior enrichment. The results showed that the modifications of the Npropenal-type at α- , as well as the DHP-type at the α-N-terminal valine, are significantly higher in hemoglobin isolated from the blood of smokers than in nonsmoking individuals. This is the first report to identify and quantify multiple sites of MDA-induced modifications in human hemoglobin from peripheral blood. Our results suggest that the MDA-derived modifications on hemoglobin might represent valuable biomarkers for MDA-induced protein damage.