A dynamic thiol/disulfide balance is pivotal in organizing anti-oxidant defense, detoxification, apoptosis, and enzyme activities, as well as transcription and cellular signal-transfer mechanisms. The connection between urolithiasis and oxidant/antioxidant status, which can be assessed through thiol-disulfide homeostasis (TDH), has not yet been examined. In this study, we evaluated the effects of TDH on the formation, size, and location of stones by examining the associations between TDH parameters and urolithiasis.Patients with urolithiasis and healthy controls were recruited. The patients were divided into subgroups in terms of stone size (>15 mm or ≤15 mm) and stone location (nephrolithiasis or ureterolithiasis). TDH parameters were measured using a novel automatic and spectrophotometric method and compared statistically.TDH parameters were different between the urolithiasis and control groups. TDH tended towards the disulfide side in the urolithiasis group. Stone size increased an average 0.14 mm with a 1 µmol/L increase in disulfide level and decreased an average 0.058 mm with a 1 µmol/L increase in native thiol level. Disulfide and native thiol levels were found to be different across patients with stone size >15 mm, ≤15 mm, and controls (p<0.001 and p<0.001, respectively). However, the nephrolithiasis and ureterolithiasis groups were similar in respect of TDH parameters.In this study, it was found that patients with urolithiasis displayed oxidative stress characterized by a TDH tendency towards the disulfide side, and an inadequate antioxidant response identified by a lower level of native thiol as compared with healthy controls.