Black soybeans are rich in isoflavones, which have several beneficial health effects. In this study, a validated method based on UHPLC-MS/MS was developed to screen black-soybean metabolites in rat urine, bile, and plasma and to quantify the compounds (daidzein, genistein, glycitein, and daidzin) and their metabolites (daidzein-4'-beta-D-glucuronide, genistein-7-beta-D-glucuronide, and genistein-4'-beta-D-glucuronide) in plasma. Thirty-seven compounds were tentatively detected in the biological samples. The method was fully validated in quantitative experiments, including in assessments of linearity (2.5-100 ng/mL for daidzein, genistein, and glycitein; 10-100 ng/mL for daidzin; 5-3125 ng/mL for genistein-7-beta-D-glucuronide; and 5-1562.5 ng/mL for daidzein-4'-beta-D-glucuronide and genistein-4'-beta-D-glucuronide), matrix effects (85-115%), recovery (80-105%), precision (<10%), and accuracy (<10%). The compounds were stable throughout sample storage, treatment, and analysis. The method was first applied to detect IFs and metabolites in rats after oral administration of black-soybean extract. These results support the potential of this method for successful application in pharmacokinetic studies.