The infrared spectroscopy and dynamics of -CO labels in wild type and mutant insulin monomer and dimer are characterized from molecular dynamics simulations using validated force fields. It is found that the spectroscopy of monomeric and dimeric forms in the region of the amide-I vibration differs for residues B24-B26 and D24-D26, which are involved in dimerization of the hormone. Also, the spectroscopic signatures change for mutations at position B24 from phenylalanine, which is conserved in many organisms and is known to play a central role in insulin aggregation, to alanine or glycine. Using three different methods to determine the frequency trajectories (solving the nuclear Schrödinger equation on an effective 1-dimensional potential energy curve, using instantaneous normal modes, and using parametrized frequency maps) leads to the same overall conclusions. The spectroscopic response of monomeric WT and mutant insulin differs from that of their respective dimers, and the spectroscopy of the two monomers in the dimer is also not identical. For the WT and F24A and F24G monomers, spectroscopic shifts are found to be ∼20 cm-1 for residues (B24-B26) located at the dimerization interface. Although the crystal structure of the dimer is that of a symmetric homodimer, dynamically the two monomers are not equivalent on the nanosecond time scale. Together with earlier work on the thermodynamic stability of the WT and the same mutants, it is concluded that combining computational and experimental infrared spectroscopy provides a potentially powerful way to characterize the aggregation state and dimerization energy of modified insulins. © 2020 American Chemical Society. All rights reserved.