Soybean rust, which is a devastating foliar disease caused by Phakopsora pachyrhizi, has spread to most soybean growing regions of the world. In this study, proteomic approach was used to investigate protein expression profile of rust-infected and uninfected (control) soybean leaves. Leaves of two 'rust susceptible cultivars, DTl2 and Vang Muong Khuong (VMK), were infected. After nine days infection, total protein was extracted from the leaves and separated by Two-Dimensional Polyacrylamide Gel Electrophoresis (2DE). The SDS-PAGE analysis did not reveal differences in levels of protein expression between the test and control samples. However, more than 120 protein spots were detected from DT12 and VMK when the 2DE gel image was analysed using Progenesis SameSpots. Of these protein spots, eight (1, 2, 3, 4, 5, 5, 6, 7 and 9) were upregulated and one (spot 8) down-regulated when the control and test samples of cultivars DTl2 and VMK were compared. For identification, there were 6 characterized proteins using nano LC connected to ESI Q TRAP mass spectrometry. Of these, there were 5 increased proteins and 1 decreased protein. Besides, three spots could not be characterized in the protein data. Further research is needed to identify and predict the role and function of the protein changes related to disease resistance of plants. These changes might playa core role in soybean rust infection. The above results indicated that the proteomic approach could be a powerful method for protein analysis, especially proteins related to diseases.