Infrared-active vibrational (IRAV) modes are specific optical fingerprints to probe the density, dynamics, and spatial distribution of polarons in π-electron conjugated polymers. So far, the description of IRAV mode activation and selection rules, resulting from the local breaking of spatial symmetry induced by charge carriers, has been restricted to phenomenological lattice-dynamics models. Overcoming the classical picture, here we combine first-principles calculations with vibrational spectroscopy to study the nuclear dynamics of a model polymer system, poly(3-hexylthiophene) (P3HT). We assign and reproduce quantitatively the transition energies and intensities of vibrational normal modes in the ground and excited electronic states. By comparing the ground, polaronic, and excitonic states of regioregular (RR-) and regiorandom (RRa-) chains, we identify, for the first time, the vibrational fingerprints of neutral singlet excitations in the IRAV spectra of P3HT and highlight structure–property correlations. Within this new approach, vibrational spectroscopy provides a comprehensive tool to study not only polaron but also exciton density and dynamics and to better understand the influence of disorder on exciton and charge-carrier localization in functional organic systems.