Drought stress severely limits crop productivity and the expansion of crop cultivation worldwide. Plants can react and adjust to water stress by shifting their cellular metabolism and invoking various defence mechanisms, and acquired stress tolerance in plants is often a result of various stress-response mechanisms that act co-ordinately or synergistically to avert cell damage and to re-establish cellular homeostasis. Metabolic adaptation via the de-novo synthesis of glycinebetaine or proline is often regarded as a basic strategy for the protection and survival of plants under drought stress. In the present study, we investigated the biochemical mechanisms of proline and betaine mediated drought tolerance in mustard (Brassica juncea L.) seedlings. Before imposition of drought stress, seedlings were fed with exogenous proline and betaine (1 mM, 24 h). Betaine or proline pre-treatment resulted in enhanced oxidative stress tolerance, as indicated by greatly reduced levels of lipid peroxidation. Endogenous hydrogen peroxide levels in proline or betaine pre-treated droughtstressed seedlings were significantly lower in comparision to seedlings exposed to drought stress without pre-treatment. A significant decrease in ascorbate peroxidase, catalase and glyoxalase II activities was found in response to drought stress, whereas dehydroascorbate reductase, glutathione peroxidase and glyoxalase I activities increased significantly. The levels of ascorbate, glutathione and the size of the glutathione disulphide pool increased significantly whereas the glutathione/glutathione disulphide ratio decreased in seedlings treated with drought stress. Importantly, drought-stressed seedlings pre-treated with betaine or proline showed significantly higher ascorbate peroxidase, glutathione reductase, catalase, glutathione S-transferase, and glyoxalase II activities and a higher glutathione/glutathione disulphide ratio than that of the seedlings imposed to drought stress without pre-treatment. This study proved that pre-treatment of seedlings with proline or betaine can modulate the methylglyoxal and reactive oxygen species levels and increase plant tolerance to drought-induced oxidative stress.