Aim: The present study was undertaken to screen wheat cultivars for resistance against stripe rust and the improvement of resistance by exogenous gamma-aminobutyric acid (GABA) under field conditions and studying two contrasting cultivars for antioxidant related defense activities at 1, 2 and 4 d of GABA, Puccinia striiformis f. sp. tritici (Pst) and GABA+Pst treatments given to one-month-old seedlings. Methodology: Wheat cultivars (PBW723, PBW725, PBW677, PBW550, PBW343, PBW621, HD2967) were grown and exposed to Puccinia striiformis f. sp. tritici (Pst) and GABA+Pst in fields and studied for yield parameters. Subsequently, two cultivars found varying in resistance (PBW725 and HD2967) were analyzed for antioxidant enzymes in leaves and roots at 1, 2, and 4 day of GABA, Pst, and GABA+Pst and water as control given to 30 days old seedlings in the greenhouse. Results: Field analysis found that the most resistant cultivar PBW725 reduced vegetative growth but not yield compared to other resistant cultivars (PBW723 and PBW677) under Pst and improved both growth and yield under GABA+Pst. GABA+Pst improved yield parameters in other cultivars also. In defense analysis, superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione-S-transferase (GST) increased in the leaves of PBW725 while only APX increased in HD2967 at 1 d of Pst. In roots also, SOD and GST increased 2-fold in PBW725 while only GST increased 1.3-fold in HD2967 at 1 d of Pst. There was persistence of catalase (CAT), monodehydroascorbate reductase (MDHAR), and dehydroascorbate reductase (DHAR) during Pst in the leaves of PBW 725 while CAT decreased at 4 d in HD2967. In roots also, CAT and MDHAR increased at 4 days of Pstin PBW725 while DHAR decreased in HD2967. Under GABA, cultivar differences were not significant. GABA+Pst improved deficient antioxidant enzymes in HD2967. Interpretation: Energy conservation and timely regulation of antioxidant enzymes during infection was utilized by PBW725 against stripe rust. GABA deficiency can be a reason for poor regulation of antioxidants in HD2967 under Pst. GABA application improved resistance against stripe rust in wheat. Key words: Antioxidant, Ascorbate, Gamma-aminobutyric acid, Glutathione, Stripe rust, Triticum aestivum