Drought is one of the environmental stressors that limits plant growth, production, and is a great threat to the rising population and security throughout the globe. In the present work, we aimed to check the impact of drought stress given at different days interval drought (DID) on proso millet and its tolerance capability. The pot culture experiment was conducted and drought stress was given at 3 DID, 5 DID, and 7 DID respectively, and sampling was done on 15, 30, and 60 DAS, and unstressed plants were irrigated regularly. Drought stress results in decreased protein contents in both the roots and shoots of P. miliaceum on all sampling days. However, with increasing drought intensity and duration there was a tremendous increase in amino acids, and proline contents as well. Therefore, increasing osmolytes like proline with an increased drought period proves the role of proline in stress tolerance of proso millet and helps it to survive by maintaining osmotic potential, water influx, and detoxifying ROS. Thus, proso millet can be cultivated in drought-prone areas, and used for the breeding program