Abstract Our laboratory treats guinea pigs with hyperbaric oxygen (HBO) as a model for investigating the formation of nuclear cataract. Previous analyses of lens supernatants using this model have shown an increase in disulfide (–SS–) and loss of sulfhydryl (–SH) in the lens nucleus of O 2-treated animals. In this paper, we have used the non-invasive technique of Raman spectroscopy to confirm these findings in intact, freshly-excised lenses. Guinea pigs were treated 3 times per week with HBO for a total of 50 (4 months of treatment) or 85 (7 months of treatment) times to induce an increased level of lens nuclear light scattering. Intact lenses were analyzed by Raman spectroscopy using a 514.5 nm laser and collecting the scattered light in a 90° geometry. The laser beam was focused either in the lens nucleus or equatorial cortex. Changes in the levels of –SS– (503 cm −1) and –SH (2577 cm −1) vibrations were measured. Raman spectra were analyzed by fitting Lorentzian profiles to the observed data in the –SS– and –SH regions. –SS– levels in the O 2-treated nucleus were found to have increased by a factor of 2.1 ( p = 0.0001) and 2.5 ( p = 0.001) after 50 and 85 HBO treatments, respectively, compared to age-matched controls. Based on previous biochemical analyses, the –SS– increase was due mainly to the formation of protein disulfide (PSSP) with contribution also from protein/thiol mixed disulfides, but not from oxidized glutathione. –SH levels in the O 2-treated nucleus decreased by 13% ( p = 0.007) and 35% ( p = 0.001) after 50 and 85 HBO treatments, respectively, compared to age-matched controls. No significant increase in –SS– or loss of –SH was observed in the lens cortex of the O 2-treated guinea pigs. The Raman spectroscopy results rule out the possibility that artifactual production of –SS– and loss of –SH occurred during homogenization of lenses in previous studies. The data provide additional evidence to support a link between O 2, disulfide-crosslinking of lens crystallins in the nucleus, and nuclear cataract.