Diagnosis of intraocular lymphoma is difficult. Among the hurdles in the diagnosis are the variety of reactive inflammatory and ischemic changes among intraocular lymphoma patients. Thus, a novel diagnostic method is desired such that lymphoma cells can be distinguished by the signals intrinsic to the cells, not by those from the surrounding tissues with reactive changes. Raman spectroscopy is a technique that can detect intrinsic signals from each cell. Therefore, Raman spectroscopy is a good candidate for an intraocular evaluation technology that could contribute to improve the diagnosis of intraocular lymphoma. In this study, we tested whether the intrinsic Raman signals from malignant lymphoma cells, in the absence of surrounding tissue, were sufficient for the discrimination of malignant lymphoma cells from leukocytes. We acquired spectra from dissociated lymphoma cells, along with spectra from normal B cells and other leukocytes involved in intraocular inflammatory diseases. We analysed the spectra using principal component analyses and quadratic discriminant analyses. We found that Raman spectra from dissociated cells without confounding tissues showed high discriminating ability, regardless of the variation due to day-to-day differences and donor differences. The present study demonstrates the possible effectiveness of Raman spectroscopy as a tool for intraocular evaluation.