Background: There is a growing body of evidence showing that there is an association between oxidative damage and kidney injury in patients with nephrotic syndrome (NS). Dynamic thiol/disulphide homeostasis (DTDH) is an important antioxidant system. The aim of this study was to determine if DTDH could be used as a biomarker of antioxidant status in pediatric NS patients using a novel automated method. Methods: The study included 39 patients with NS and 40 healthy controls. The DTDH level was measured using a calorimetric and automated method developed by Erel and Neselioglu. In the NS group, DTDH was measured during first-attack episode, relapse, and remission. Results: The native thiol, total thiol, and disulphide levels were significantly lower, the native thiol/total thiol ratio was significantly lower, and the disulphide/native thiol and disulphide/total thiol ratios were significantly higher in the NS patients during relapse and first-attack episode than during remission and in the controls. In addition, the native thiol and total thiol levels, and the native thiol/total thiol ratio were significantly lower, and the disulphide/native thiol and disulphide/total thiol ratios were significantly higher in the NS group during remission than those in the control group. Conclusions: The present findings show that DTDH shifted toward disulphide formation in the NS patients, even during remission. A clearer understanding of the mechanism of this disruption to homeostasis in NS patients might lead to the development of novel therapeutic strategies.