A novel combination of 2-D electrophoresis with hydrophobic partitioning in aqueous two-phase systems (ATPS) was extended to an alternative ATPS and both systems used for the three-dimensional characterization of the proteins extracted from soybeans. The 3-D plots of molecular weight, isoelectric point, and surface hydrophobicity were obtained using two different phase-forming salts: Na2SO4 and potassium phosphate. Six proteins with known hydrophobicities were used to validate the ATPS-based method. Molecular properties obtained using the PEG-sulfate system resulted in a wider range of proteins characterized. The wide range of concentration and strongly hydrophilic character of the soy extracts limited the coverage obtained; reduction of the storage protein content aided detection. The number of proteins simultaneously and accurately characterized by this method as currently implemented is limited by the dynamic range of staining, the ability to quantify strongly partitioning proteins in both phases, and loss of proteins of limited solubility in the ATPS or during the removal of phase-forming components.