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Differential interference contrast microscopy is designed to image unstained and transparent specimens by enhancing the contrast resulting from the Nomarski prism-effected optical path difference. Retinitis pigmentosa, one of the most common inherited retinal diseases, is characterized by progressive loss of photoreceptors. In this study, Differential interference contrast microscopy was evaluated as a new and simple application for observation of the retinal photoreceptor layer and retinitis pigmentosa diagnostics and monitoring. Retinal tissues of Royal College of Surgeons rats and retinal-degeneration mice, both well-established animal models for the disease, were prepared as flatmounts and histological sections representing different elapsed times since the occurrence of the disease. Under the microscopy, the retinal flatmounts showed that the mosaic pattern of the photoreceptor layer was irregular and partly collapsed at the early stage of retinitis pigmentosa, and, by the advanced stage, amorphous. The histological sections, similarly, showed thinning of the photoreceptor layer at the early stage and loss of the outer nuclear layer by the advanced stage. To count and compare the number of photoreceptors in the normal and early-retinitis pigmentosa-stage tissues, an automated cell-counting program designed with MATLAB, a numerical computing language, using a morphological reconstruction method, was applied to the differential interference contrast microscopic images. The number of cells significantly decreased, on average, from 282 to 143 cells for the Royal College of Surgeons rats and from 255 to 170 for the retinal-degeneration mouse. We successfully demonstrated the potential of the differential interference contrast microscopy technique's application to the diagnosis and monitoring of RP.