A perfusion RP HPLC method enabling the separation of soybean proteins in an analysis time lower than 3 min has been used to obtain the chromatographic profiles of different soybean cultivars. The chromatograms obtained for each soybean variety presented clear differences that justified the potential use of this method for cultivar characterization. The area percentages obtained were employed as variables for cluster and principal components analysis of these soybeans. The application of these multivariate methods enabled the grouping of the soybeans in different categories. The protein fractions obtained from these soybeans by the application of a fractionation method were also analyzed. The chromatographic profiles obtained enabled the assignment of peaks to the main soybean proteins (7S and 11S globulins). These data were used for the estimation, for the first time, of the 7S and 11S globulin contents in soybean cultivars.