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Grass pea (Lathyrus sativus L.) is the most drought-tolerant legume crop rich in dietary protein.However, little is known about the molecular mechanisms of its drought resistance. Two-dimensional gel electrophoresis (2-DE) is an important experiment technique in proteomics, which has been applied extensively in studies on plant resistance to abiotic stress. To establish an effective 2-DE platform and further study the droughtresistance mechanisms of grass pea using proteomic approaches, three protein extraction methods, different isoelectric focusing (IEF) conditions and various types of gel strips were evaluated using mature leaves. The results showed that the trichloroacetic acid (TCA)/acetone protein extraction method, extending time at low voltage for IEF and using 18 cm gel strip with pH 4.0-7.0 were optimum conditions for 2-DE analysis of grass pea leaves. Applying these optimized 2-DE conditions, 1,481 total protein spots were detected in control leaves and 1,346 spots in polyethylene glycol -treated leaves, of which 67 differentially expressed protein spots were obtained relative to the control. These data suggested that an efficient 2-DE platform with high repeatability and resolution for grass pea mature leaves had been established for the first time here, which could be further used to investigate the droughtresistance molecular mechanisms of grass pea.