The present study deals with the scientific evaluation of antioxidant potential of aqueous extract of Trichosanthes dioica fruits on diabetes-induced oxidative stress of diabetic rats. The most effective dose of mg/kg bw of fruit aqueous extract was given orally to diabetic rats for 30 days. Different oxidative stress parameters were analyzed in various tissues of control and treated diabetic rats. The observed elevated level of lipid peroxidation (LPO) comes down significantly (p < 0.05) and decreased activities of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione-S-transferase (GST) got increased (p < 0.05) significantly of diabetic rats on extract treatment. Laser-Induced Breakdown Spectroscopy (LIBS) has been used as an analytical tool to detect major and minor elements like Mg, Fe, Na, K, Zn, Ca, H, O, C, and N present in the extract. The higher concentration of Ca(2+), Mg(2+), and Fe(2+), as reflected by their intensities are responsible for antioxidant potential of T. dioica.