Drought tolerant pearl millet genotype was used for diff erential physiological and proteomic analysis. The water stress was imposed on 20 days seedling up to fi ve days. The physiological parameters viz. soil moisture content, relative water content (RWC), shoot length (cm) were studied from drought and control seedling a er 25 days. The results showed signifi cant changes on RWC and soil moisture content was decreased under water stress. Proteome analysis of 2D gel electrophoresis indicates around 1262 well resolved spots within the 4-7 pH and 10-110 kDa ranges. Image analysis revealed the presence of both, qualitative and quantitative changes between two treatments. The proteomic changes were observed in tolerance genotype J-2340 resulted total 84 spots protein (22.5-97.4 kDa, pH- 4.00 to 6.73) matches with control and water stress treatments. However, 32 proteins up regulated (29.0-97.4 kDa, pH 4.20-7.00) and eight down regulated (57.9-97.4kDa, pH 4.00 to 6.68) were observed a er imposing water stress.