A xanthene derivative, Granada Green dinitrobenzene sulfonate (GGDNBS), has been synthesized to assay cellular oxidative stress based on changes in the concentration of biothiols. The dye is able to react with biological thiols by a thiolysis reaction that promotes a change in fluorescence intensity. To demonstrate the usefulness of GGDNBS for in vivo oxidative stress measurements, 661 W photoreceptor-derived cells were exposed to light to induce ROS generation, and changes in GGDNBS fluorescence were measured. In these cells, GGDNBS fluorescence was correlated with the biothiol levels measured by an enzymatic method. Therefore, GGDNBS allows us to monitor changes in the levels of biothiols associated with ROS generation via single-cell bioimaging.