© 2018 Elsevier Ltd β-Conglycinin, a major protein in soybeans, shows improvement effect of lipid metabolism. Moreover, this protein influences the processing properties of soybeans. β-Conglycinin is a hetero-trimer constituted by α α′ and β subunits. In this work, a method for the selective quantification of these subunits was developed by means of protein absolute quantification (AQUA) technology using liquid chromatography/tandem mass spectrometry with the stable isotope-labelled internal standard peptides LQSGDALR[13C6,15N4], NILEASYDTK[13C6,15N2], and NPIYSNNFGK[13C6,15N2]. This method exhibited linear relationships (r2 > 0.99) in the concentration range of 1.2–300 fmol/μL for LQSGDALR[13C6,15N4] and NILEASYDTK[13C6,15N2], and of 4.7–300 fmol/μL for NPIYSNNFGK[13C6,15N2]. As a result, the content of these subunits in β-conglycinin-rich and both α and α′ subunit-deficient soybean cultivars was successfully determined. This quantitative assay is promising for the evaluation of the food functionality and processing properties of soybeans.