Exposure to excessive light induces retinal photoreceptor cell damage, which may involve lipid peroxidation. Morphological changes and the detection of internucleosomal DNA fragmentation confirmed the retinal damage caused by exposure of the retina of Balb/c mice to white fluorescent light (5000 lux, 2 h). The total amounts of hydroxyoctadecadienoic acid (tHODE) and 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha) in the retinas obtained from light-exposed mice were assessed after reduction and saponification. In this method, both the free and ester forms of hydroperoxides, hydroxides, and ketones of linoleic acid are measured as tHODE by gas chromatography-mass spectrometry (GC-MS) analysis. When compared with controls, a significant increase in the concentrations of tHODE and 8-iso-PGF2alpha was observed 24 h after light exposure. Furthermore, the stereoisomeric ratio (Z,E)-HODE/(E,E)-HODE decreased after light exposure, suggesting the involvement of free-radical-mediated peroxidation. By the immunohistochemical technique, it was confirmed that 8-iso-PGF2alpha increased in the inner plexiform layer (IPL), outer plexiform layer (OPL), rod outer segment, and choroidal layer, while 13-HODE increased in the OPL and rod inner segment after light exposure. These results demonstrate that tHODE and 8-iso-PGF2alpha assessed by the present method are appropriate biomarkers responding to retinal photooxidative stress in vivo.