Background: Thiol/disulphide homeostasis plays a critical role in numerous intracellular enzymatic pathways including antioxidant defense and detoxification. This study was designed to investigate the impact of thiol/disulfide homeostasis in adolescent patients with recently diagnosed primary hypertension (HT) using a novel and automated method. Methods: Native thiol/disulphide levels were measured by a novel spectrophotometric method (Cobasc 501, Roche Diagnostics, Mannheim, Germany) in 30 patients with primary HT together with 30 healthy controls. Results: The levels of native thiol, total thiol, and native thiol/total thiol ratios were significantly lower, while the disulphide level, disulphide/native thiol, and disulphide/total thiol ratios were significantly higher in patients with primary HT compared with the control group. There were significant positive correlations between 24-h mean systolic and diastolic blood pressure and disulphide levels, disulphide/native thiol, and disulphide/total thiol ratios. A multiple linear regression model showed that a disulphide/native thiol ratio above 5 and family history of HT are independent predictors of HT. Conclusions: Our study showed that dynamic thiol/disulphide homeostasis shifted towards disulphide formation in adolescent patients with primary HT. Understanding the role of thiol/disulfide homeostasis in primary HT might provide new therapeutic intervention strategies for patients.