Hemoglobin (Hb) adducts have been used as biomarkers for the internal exposure to chemicals. Simultaneous exposure to chemicals that bond with the N-terminal valine of Hb to form adducts, such as glycidol, acrylamide, and glucose, may affect the formation of the individual Hb adducts. In this study, various factors influencing the formation of chemical–Hb adducts were analyzed using in vitro and in vivo systems. In the in vitro assays, the formation of glycidol– and acrylamide–Hb adducts was altered in the presence of glucose, serum albumin, and other chemicals. In contrast, in the in vivo experiments, glycidol– and acrylamide–Hb adduct formation was unchanged in mice exposed to glycidol and acrylamide. The interaction between glycidol and acrylamide with residues other than the N-terminal valine of Hb was analyzed using the protein thermal shift assay. Glycidol and acrylamide also interacted with amino acid residues other than the N-terminal valine of Hb. The presence of other blood components, such as amino acids, may affect the formation of chemical–Hb adducts. Further research is expected to elucidate the remaining unknown factors that affect the formation of chemical–Hb adducts.