Drought stress severely limits plant growth and development, however, the molecular and genetic basis of sorghum (Sorghum bicolor (L.) Moench) response to drought stress is poorly understood. To unravel the molecular mechanism of drought tolerance of sorghum, comparative proteomics analysis was performed on drought-tolerant variety JTY and its EMS drought-sensitive mutant E24 seedlings treated with 25% PEG 6000 simulated drought stress for 24 hours. Over 3000 proteins were accurately quantified from four samples. KEGG analysis showed that after PEG treatment, the pathways related to carbohydrate metabolism were affected, Starch synthase was decreased, while plant-pathogen interaction and phenylpropanoid biosynthesis were significantly enhanced under PEG stress. Ribosome, Biosynthesis of amino acids, Carbon metabolism and Photosynthesis were common enriched by mutant related DAPs and mutant response to PEG stress. some stress and mutant related DAPs were verified by parallel reaction monitoring (PRM) and qRT-PCR, respectively. Our findings offer insights into the molecular networks mediating sorghum response to drought stress.