Dietary soybean saponins have been proposed the potential role in plasma cholesterol-lowering, colon cancer inhibiting and immune-stimulating effects to human health. To evaluate the biological properties of soybean saponins, saponin concentrations in soy products must be determined. This has led to the need for sensitive and accurate assays of saponins for food matrices. Methods for isolation and quantitative determination of the group B soybean saponins by high performance liquid chromatography (HPLC) were developed. 2, 3-Dihydro- 2, 5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP)-conjugated soybean saponin αg, βg, βa, and their non-DDMP counterparts I, II, and V were isolated and identified by electrospray mass spectrometry and ultraviolet absorbance spectrometry in HPLC profile. The yield of purified soyasaponin was > 0.017% (w/w), and the purity was > 95%. The molar extinction coefficients for the group B saponins were ranged from 4500 to 6000 M−1cm−1 at 292 and 205 nm. The purified DDMP-conjugated saponin standards were stable at -200C for at least 15 days or at 300C for 3 hours without observable decomposition. The coefficient of variation of saponin quantitation analysis by HPLC was 9.8% for the within-day assay and <14.3% for the between-days assay. The external recovery of saponin analysis showed >93% recovery for soyasaponin I. Formononetin was demonstrated to be a good internal standard in soyasaponin analysis. The soyasaponin contents were evaluated in soybeans and soy products. The group B saponin concentration in soybeans ranged from 2.50 to 5.85 [Mu]mol/g and varied among different varieties of soybeans. The saponin content and composition in soy ingredients (soybean seeds, toasted soy hypocotyls, soy protein isolates, textured vegetable protein, soy protein concentrates, and Novasoy[Registered Trademark symbol]), and the typical soy foods (commercial soymilk, tofu, and tempeh) were ranged from 0.47 to 114.02 [Mu]mol/g. In the commercial soy products, the ratio of total isoflavone to soyasponin on a molar basis was ranged from 0.2 to 20.4 depending on the production process.