Fluorogenic probes for redox metabolism are shown to exhibit two-photon absorption using femtosecond pulses from a Ti/sapphire laser. The probes consist of a coumarin or naphthalene core and a ketone−alcohol functional group. The probe design is based on the changes in fluorescence properties and two-photon cross section values when the ketone derivative is reduced to the corresponding alcohol. The resulting contrast ratio of the fluorescence differs significantly from that obtained by one-photon excitation. This phenomenon was demonstrated with all three switches examined herein and represents an attractive approach to modulation of emission properties of molecular switches. The practical applicability of the two-photon-excitation redox switch was demonstrated in an enzyme-catalyzed transformation. The nonfluorescent probe 1is efficiently converted by AKR1C3, a human 3-hydroxysteroid dehydrogenase, to the fluorescent derivative 2. Using two-photon excitation (775 nm), we achieved a large contrast ratio between the fluorescence in the presence and the absence of the enzyme of ∼300.