Variations in the proportions of the two major soybean [Glycine max (L.) Merr.] seed globulins, glycinin (11S) and β-conglycinin (7S), significantly affect the nutritional and functional properties of soy-based products, but comprehensive methods for the identification and quantification of individual subunits of these proteins are currently lacking. We developed an optimized reverse-phase ultra-performance liquid chromatography (RP-UPLC) method to analyze 11S and 7S protein contents in the seeds of three soybean varieties grown in different years. Using commercial protein standards and subunit-null varieties, we successfully identified and quantified all 11S and 7S protein subunits in Williams 82, Daepung, and Kwangan. The 11S + 7S proteins accounted for 72.6-76.2 %, 61.9-67.2 %, and 65.8-80.7 % of total proteins from these varieties (depending on cultivation year), with 11S/7S ratios of 1.82-2.28, 1.79-2.03, and 2.18-2.75, respectively. This RP-UPLC method is valuable for studying the physiochemical properties of soy-based products and selecting desirable varieties.