We investigated serum and aqueous humor thiol/disulfide (T-D) homeostasis in patients with cataracts versus healthy controls. In total, 56 patients with cataracts and 49 healthy controls were enrolled in this case-control study. Serum total thiol (TT), native thiol (NT), and disulfide (DS) concentrations were determined using a novel automated measurement method. Additionally, DS/TT, DS/NT and NT/TT percentage ratios were compared between the groups. In comparison with the control group, serum NT levels and aqueous humor TT and NT levels were significantly lower (p<.05, p<.05 and p<.001, respectively), whereas serum and aqueous humor DS levels were significantly higher in cataract patients (p<.01 and p<.001). DS/TT and DS/NT ratios were significantly higher and the NT/TT ratio was lower in cataract patients in serum (p<.005) and aqueous humor samples (p<.001). In conclusion, serum T-D homeostasis may be useful as biochemical markers, indicating the role of oxidative stress in the development of cataracts. Further studies are needed to confirm the pathophysiological role of T-D homeostasis in cataractogenesis.