PURPOSE. An often overlooked prerequisite to cone photoreceptor gene therapy development is residual photoreceptor structure that can be rescued. While advances in adaptive optics (AO) retinal imaging have recently enabled direct visualization of individual cone and rod photoreceptors in the living human retina, these techniques largely detect strongly directionally-backscattered (waveguided) light from normal intact photoreceptors. This represents a major limitation in using existing AO imaging to quantify structure of remnant cones in degenerating retina. METHODS. Photoreceptor inner segment structure was assessed with a novel AO scanning light ophthalmoscopy (AOSLO) differential phase technique, that we termed nonconfocal splitdetector, in two healthy subjects and four subjects with achromatopsia. Ex vivo preparations of five healthy donor eyes were analyzed for comparison of inner segment diameter to that measured in vivo with split-detector AOSLO. RESULTS. Nonconfocal split-detector AOSLO reveals the photoreceptor inner segment with or without the presence of a waveguiding outer segment. The diameter of inner segments measured in vivo is in good agreement with histology. A substantial number of foveal and parafoveal cone photoreceptors with apparently intact inner segments were identified in patients with the inherited disease achromatopsia. CONCLUSIONS. The application of nonconfocal split-detector to emerging human gene therapy trials will improve the potential of therapeutic success, by identifying patients with sufficient retained photoreceptor structure to benefit the most from intervention. Additionally, splitdetector imaging may be useful for studies of other retinal degenerations such as AMD, retinitis pigmentosa, and choroideremia where the outer segment is lost before the remainder of the photoreceptor cell. © 2014 The Association for Research in Vision and Ophthalmology, Inc.