Among many other functions, flavin serves as a chromophore in LOV (light-, oxygen-, or voltage-sensitive) domains of blue light sensors. These sensors regulate central responses in many organisms such as the growth of plants towards light. The triplet-excited state of flavin (³Fl) has been identified as a key intermediate in the photocycle of LOV domains, either in its neutral or protonated state. Even time-resolved infrared spectroscopy could not resolve unambiguously whether ³Fl becomes protonated during the photoreaction, because the protonated triplet-excited state ³FlH⁺ has not been characterized before. Here, the step-scan Fourier transform infrared (FTIR) technique was applied to the flavin mononucleotide (FMN) in aqueous solution at different pH values to resolve laser-induced changes in the time range from 1.5 μs to 860 μs. A high-pressure-resistant flow cell system was established to account for the irreversibility of the photoreaction and the small path length. Several marker bands were identified in the spectrum of ³Fl in water and assigned by quantum chemical calculations. These bands exhibit a solvent-induced shift as compared with previous spectra of ³Fl in organic solvents. The marker bands undergo a further distinct shift upon formation of ³FlH⁺. Band patterns can be clearly separated from those of the anion radical or the fully reduced state resolved in the presence of an electron donor. A comparison to spectra of ³Fl in LOV domains leads to the conclusion that ³FlH⁺ is not formed in the photoreaction of these blue light sensors.