The two-dimensional infrared spectra of a series of doubly isotopically substituted 25-residue α-helices were measured with femtosecond three pulse infrared time domain interferometry. The insertion of 13C16O and 13C18O labels at known residues on the helix permitted the vibrational couplings between different amide I‘ modes separated by one, two, and three residues to be measured. The 2D IR signal of one residue in 25 was readily studied, confirming this approach is applicable to labeled proteins. We identified the couplings between each pair of isotopomer levels and between them and the helix exciton band states: the 2D IR spectra proved that the amide vibrations of the α-helix are delocalized. Cross-peaks, originating from the coupling of the isotopomer pairs, were systematically analyzed. Besides the separated pair modeling and second-order perturbation theory estimates, the experimental results were compared in detail with a full matrix diagonalization simulation based on averaged Hamiltonian matrices that represent the amide I‘ vibrator's one- and two-exciton states. The main features of the 2D IR spectra could be predicted by this modeling. The experimental results were in good agreement with a set of couplings that were derived from transition charge−transition charge interactions for all but the nearest neighbors, for which the coupling is more influenced by through-bond interactions between the adjacent amide groups. The possible ranges of the magnitudes of the three largest coupling constants β12, β13, and β14 were explored by various approaches to be within a few cm-1 accuracy of a preferred set of absolute values and their associated error bars: |β12| = 8.5 ± 1.8, |β13| = 5.4 ± 1.0, and |β14| = 6.6 ± 0.8 cm-1. The signs were independently indicated to be β12 > 0, β13 < 0, and β14 < 0.