The protonation state of ionizable residues is an important contributor to protein stability and function. Histidine is of particular importance because its side chain pK(a), is near physiological pH. The sensitivity of carbon deuterium (C-D) vibrational frequencies to the protonation state of histidine dipeptide (Hdp) was investigated in the gas-phase using density functional theory (DFT) calculations, and in aqueous solution using two-layered integrated molecular orbital and molecular mechanics (ONIOM) calculations. All three C-D vibrational probes on the side chain (C-beta-D-2, C-delta-D, and C-epsilon-D) independently exhibited a striking sensitivity to the gas-phase histidine protonation state, with calculated shifts of up to 40 cm(-1) upon deprotonation of the histidine residue. Simultaneously including all three C-D vibrational probes on the Hdp side chain produced significant shifts of 2.8 to 43 cm(-1) between the neutral and charged states. The calculated intensities also dropped precipitously upon deprotonation, which is an important factor for the interpretation of experiments employing C-D vibrational probes to investigate side chain protonation. Solvating the labeled Hdp molecule produces an overall blue-shift in the average C-D vibrational frequencies relative to the gas-phase. The C-beta-D-2, C-delta-D, and C-epsilon-D vibrational probes all showed sensitivity to the histidine protonation state, with shifts of up to 40 cm(-1) in the mean frequencies after deprotonation, which bodes well for studies employing C-D probes to study histidine protonation state in peptides and proteins.