This article demonstrates experimental proof of excited state ‘solvent-to-chromophore’ proton transfer (ESPT) in the isolated gas phase PBI (2,2′-pyridylbenzimidazole)-CH3OH complex, aided by computational calculations. The binary complexes of PBI with CH3OH/CH3OD were produced in a supersonic jet-cooled molecular beam and the energy barrier of the photo-excited process was determined using resonant two-colour two-photon ionization spectroscopy (R2PI). The ESPT process in the PBI-CH3OH complex was confirmed by the disappearance of the Franck-Condon active vibrational transitions above 000 + 390 cm−1. In the PBI-CH3OD complex, the reappearance of the Franck-Condon transitions till 000 + 800 cm−1 confirmed the elevation of the ESPT barrier upon isotopic substitution due to the lowering of the zero-point vibrational energy. The ESPT energy barrier in PBI-CH3OH was bracketed as 410 ± 20 cm−1 (4.91 ± 0.23 kJ mol−1) by comparing the spectra of PBI-CH3OH and PBI-CH3OD. The solvent-to-chromophore proton transfer was confirmed based on the significantly decreased quantum tunnelling of the solvent proton in the PBI-CH3OD complex. The computational investigation resulted in an energy barrier of 6.0 kJ mol−1 for the ESPT reaction in the PBI-CH3OH complex, showing excellent agreement with the experimental value. Overall, the excited state reaction progressed through an intersection of ππ* and nπ* states before being deactivated to the ground state via internal conversion. The present investigation reveals a novel reaction pathway for the deactivation mechanism of the photo-excited N-containing biomolecules in the presence of protic-solvents. © 2023 The Royal Society of Chemistry