The pH dependent behavior of two flavin cofactors, flavin-adenine dinucleotide (FAD) and flavin mononucleotide (FMN), has been characterized using femtosecond transient absorption spectroscopy for the first time. The flavin excited state was characterized in three states of protonation (Fl(-), Fl, and FlH(+)). We found that Fl and Fl(-) exhibit the same excited state absorption but that the lifetime of Fl(-) is much shorter than that of Fl. The transient absorption spectrum of FlH(+) is significantly different from Fl and Fl(-), suggesting that the electronic properties of the flavin chromophore become appreciably modified by protonation. We further studied the excited state protonation of the flavin and found that the protonation sites of the flavin in the ground and excited state are not equivalent. In the case of FAD, its excited state dynamics are controlled by the two conformations it adopts. At low and high pH, FAD adopts an "open" conformation and behaves the same as FMN. In a neutral pH range, FAD undergoes a fast excited state deactivation due to the "stacked" conformer. The transition from stacked to open conformer occurs at pH ~ 3 (because of adenine protonation) and pH ~ 10 (because of flavin deprotonation).