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 ((3)Fl) 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 (3)Fl becomes protonated during the photoreaction, because the protonated triplet-excited state (3)FlH(+) 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 (3)Fl in water and assigned by quantum chemical calculations. These bands exhibit a solvent-induced shift as compared with previous spectra of (3)Fl in organic solvents. The marker bands undergo a further distinct shift upon formation of (3)FlH(+). 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 (3)Fl in LOV domains leads to the conclusion that (3)FlH(+) is not formed in the photoreaction of these blue light sensors.