Lead (Pb) toxicity is a major problem in agricultural soil that negatively affects plant growth and development. Glycine betaine (GB) is an effective compatible solute that resists abiotic stress and plays an important role to mitigate various stresses. The present study is the first of its kind on the application of GB to mitigate Pb toxicity on barley cultivars. To elucidate the role of GB in mitigating Pb toxicity of three concentrations (15 mM, 25 mM, and 35 mM) in two barley varieties (BH-959 and BH-946) with and without foliar application of GB (2 mM) was examined. The study found that increasing Pb concentration significantly (p<0.05) reduced the level of primary metabolites viz. photosynthetic pigments, protein, and carbohydrates in both cultivars upto 42.4%, 38.4% and 39% respectively. However malondialdehyde content, proline content, and antioxidant enzyme activity (SOD, CAT, and POX) were found to increased significantly (p<0.05) as compared to control treatment upto 83.4%, 83.2% and 51% respectively. In contrast, the application of GB led to significantly (p<0.05) improved physio-biochemical parameters as well as antioxidant enzyme activity (53%) and reduced oxidative stress along with malondialdehyde content (14.42%) in both varieties. An increment in these parameters revealed that exogenous application of GB (2 mM) significantly improves Pb (up to 35 mM) toxicity in barley plants and its use may be beneficial for crops susceptible to Pb toxicity to improve growth and yield.