Vertebrate vision is maintained by the retinoid (visual) cycle, a complex enzymatic pathway that operates in the retina to regenerate the visual chromophore, 11-cis-retinal, a prosthetic group of rhodopsin that undergoes activation by light. Many different mutations in genes encoding retinoid cycle proteins can cause a variety of human blinding diseases. Two-photon microscopy is an evolving, non-invasive, and repetitive imaging technology that can be used to monitor biomolecules within the vertebrate retina at a subcellular resolution. This method has the great advantage of portraying endogenous retinoid fluorophores in their native state without the need for artificial staining. Such real-time retinal imaging permits rapid evaluation not only of various stages of retinal disease in live animal models of human retinopathies but also of the outcome from intended pharmacological therapies. Two-photon microscopy offers substantial potential for early detection of age- and disease-related changes in the eye, long before clinical or pathological manifestations become apparent.