Nine Virginia soybeans grown in a single location were compared for their antioxidant properties and isoflavone profiles. The extracts were evaluated for their total phenolic contents (TPC), Oxygen Radical Absorbance Capacity (ORAC), and DPPH• radical scavenging activities. In order to evaluate efficient preparation methods for soybean antioxidants, three Virginia-grown soybeans were extracted using different extraction strategies. The extraction techniques included soxhlet extraction, conventional solvent extraction, and ultrasonic-assisted extraction (UAE) with 5 different common solvent systems including 50% and 80% aqueous acetone, 50 and 70% aqueous ethanol, and 80% aqueous methanol. The TPC in the soybean extracts and isoflavone compositions were significantly different among cultivars. Malonylgenistin was the major isoflavone in all soybean seeds, accounting for 75-83% of the total measured isoflavones. The V01-4937 variety had the highest total isoflavone and malonylgenistin contents, followed by V03-5794. The antioxidant activities of the soybean extracts were also significantly different. Overall, the V01-4937 soybean was the variety that stood out from the other tested Virginia soybeans because it had the highest TPC, ORAC value, and isoflavone contents as well as the second highest DPPH• scavenging activity. Ultrasonic treatment improved the extraction of soybean phenolics by more than 50% compared to solvent alone. The UAE with 50% aqueous acetone was the most efficient for extraction of phenolic compounds in the soybean seeds. The conventional and UAE with 70% aqueous ethanol extracts had the highest ORAC values, while the soxhlet methanol extracts had the highest DPPH• radical scavenging activities. Our results suggest that different extraction technologies have a remarkable effect on soybean antioxidant estimation and the UAE is more appropriate for soybean phenolic extraction because it is less time and solvent consuming than the conventional solvent and soxhlet extractions. The V01-4937 soybean with the highest TPC was evaluated for its antioxidant activity and isoflavone contents in an in vitro digestive system. After gastrointestinal digestion, soybean extracts contained higher TPC and ORAC values than cooked soybean (before digestion) but they were relatively low in DPPH• radical scavenging capacity. The glucosides, daidzin, genistin, and malonylgenistin showed stability during simulated digestion with 83.3 %, 59.4 %, and 10.7 % recovery, respectively. Aglycones, including daidzein and genistein, were recovered at 37 % and 73.7 %, respectively, after in vitro digestion. In this study, daidzin was the most stable and bioaccessible isoflavone determined using the in vitro digestive system. Among the aglycones, genistein was more stable and bioaccessible than daidzein after digestion. In conclusion, soybean antioxidant activities were different among cultivars and efficient extraction for TPC was found using UAE with 50% aqueous acetone. Furthermore, antioxidant activities were stable during digestion and genistein, within aglycones tested, was the most stable and bioaccessible compound following in vitro digestion. This information may provide manufacturers or researchers information required to develop food or nutraceutical products processed for better bioaccessibility of soybean bioactive components.