Drought is one of the most devastating environmental stress which affects the plant growth and yield in several ways. Grain crops including barley (Hordeum vulgare L.) are badly affected due to shortage of water all over the world. Ascorbic acid acts as an important cofactor in plant hormones biosynthesis and its exogenous application has been shown to confer tolerance against biotic and abiotic stresses. In the present research, the ability of ascorbic acid to increase drought stress tolerance was investigated in three barley genotypes, a drought-resistant (V3 = Jau-Haider 93) and two drought-sensitive (V1 = 92,044 and V2 = 92,112) grown in pots under field conditions. Two levels of water, control i.e., with adequate irrigation and drought stress with 60 % of field capacity were applied. Ascorbic acid was applied as a foliar spray (1 mM) after every 7 days interval at 3rd leaf stage. In the present study, imposition of drought stress significantly reduced the growth, physiological and biochemical parameters in all genotypes of barley. Among all the varieties, V3 showed the highest increase (18.2 mmolm-2s-1) in net photosynthetic rate when treated with ascorbic acid. Similarly, ascorbic acid increased transpiration rate in V3 with a value of 1.42, 1.45, 1.18, and 1.28 mmolm-2s-1 in control, control with ascorbic acid, drought stress, and drought stress with ascorbic acid, respectively. During the present investigation polyamines i.e., putrescene, spermidine and spermine was highest (117.22, 99.12 and 107.02 nmol cent g-1 FW, respectively) in V1 plants treated with ascorbic acid without drought stress. Proline, protein and carbohydrate contents was also increased significantly in barley by ascorbic acid application as compared to control plants. V1 showed the highest peroxidase, catalase and superoxide dismutase (17.23, 39.12, 17.22 Umg-1 of proteins, respectively) in plants treated with ascorbic acid as compared to control without ascorbic acid treatment. Ascorbic acid as foliar spray supported the plant growth under drought stress to ameliorate the adverse effects of stress by maintaining the relative water contents and osmotic adjustment by enhancing antioxidant enzymes, polyamines, proline and phenolics contents. (c) 2024 SAAB. Published by Elsevier B.V. All rights are reserved, including those for text and data mining, AI training, and similar technologies.