To determine the effect of biochar application (BIO) and exogenous spraying of methyl jasmonate (MJ) on some biochemical attributes of winter barley under withholding irrigation at the flowering stage (for two weeks), a two-year glasshouse study was carried out. The drought stress increased the electrolyte leakage by 51.7%, however addition of biochar into the soil and spraying methyl jasmonate decreased this parameter remarkabley. Moreover, drought stress increased the enzymatic activity of catalase in both years (1016% and 716% respectively in the first and the second year), super oxid dismutase in both years (1302% in the first year and 382% in the second year), proline content (256%), total soluble sugar content (366% in the first and 490% in the second year) and Malondyaldehyde (383% and 417% respectively in the first and the second year). However, due to application of biochar and methyl jasmonate, the adverse effects of drought stress on these characteristics were adjusted. The highest (9.51 gr/pot) and the lowest (4 gr/pot) grain yield was recorded as a result of combined application of 0.25% of biochar and 50 μmol of methyl jasmonate. Conclusively, it is deduced that, addition of biochar into the soil and spraying methy jasmonate can be effective in managing water shortage conditions if they are used in appropriate doses which were shown to be 0.25% of biochar and 50 μmol per liter of methyl jasmonate respectively, to optimize water use and minimize drought stress.