Switchgrass (Panicum virgatum) is a perennial crop producing deep roots thus highly tolerant to soil water deficit conditions. However, seedling establishment in field is very susceptible to prolonged and periodic drought stress. In this study, a “sandwich” system simulating a gradual water deletion process was developed. Switchgrass seedlings were subjected to a 20-day gradual drought treatment process when soil water tension was increased to 0.05 MPa (moderate drought stress) and leaf physiological properties had expressed significant alteration. Drought-induced changes in leaf proteomes were identified using the relative and absolute quantitation (iTRAQ) labeling method followed by nano-scale liquid chromatography mass spectrometry (nano-LC-MS/MS) analysis. Additionally, total leaf proteins were processed using a combinatorial library of peptide ligands to enrich for lower abundance proteins. Both total proteins and those enriched samples were analyzed to increase the coverage of the quantitative proteomics analysis. A total of 7,006 leaf proteins were identified, and 257 (4% of the leaf proteome) expressed a significant difference (P < 0.05, fold change < 0.6 or > 1.7) from the non-treated control to drought-treated conditions. Results from this study, in addition to revealing molecular responses to drought stress, provide a large number of proteins (candidate genes) that can be employed to improve switchgrass seedling growth and establishment under soil drought condition.