Metabolic monitoring at the cellular level in live tissues is important for understanding cell function, disease processes, and potential therapies. Multiphoton imaging of the relative amounts of NADH and FAD (the primary electron donor and acceptor, respectively, in the electron transport chain) provides a noninvasive method for monitoring cellular metabolic activity with high resolution in three dimensions in vivo. NADH and FAD are endogenous tissue fluorophores, and thus this method does not require exogenous stains or tissue excision. We describe the principles and protocols of multiphoton redox ratio imaging in vivo.