Objective: This study aimed to assess the estrogenic and antiestrogenic activities of five dietary supplements, commercially available for treatment of menopausal symptoms, before and after gastrointestinal digestion by employing a yeast steroid-regulated transcription system. Methods: Supplements (S) were extracted with either 80% methanol or water. Water extracts were subjected to simulated gastrointestinal (GI) digestion. Estrogenic and antiestrogenic activities were assessed by a steroid-regulated transcription system in Saccharomyces cerevisiae expressing the human estrogen receptor alpha. Results:The highest estrogenic activities were detected in both S1 methanol (2342.5±20.83 MU) and water (1225.6±20.6 MU) extracts (400 estradiol equivalents). Extracts showed antiestrogenic properties by reducing the transcriptional activity induced by estradiol in transgenic yeast. The highest antiestrogenic activity was detected in S2 methanol extract and S3 water extract, which inhibited estradiol activity by 76% and 64%, respectively. After GI digestion, S1, S2 and S3 extracts showed significantly higher estrogenic and antiestrogenic activities as 'serum-available' than 'colon-available' samples and S4 and S5 extracts showed significantly higher activities as 'colon-available' than 'serum-available' samples. Conclusion:All dietary supplements revealed estrogenic and antiestrogenic activities. The GI digestion demonstrated the availability of phytoestrogens for absorption in the blood stream. Supplements containing soy isoflavones and alfalfa ingredients had the highest estrogenic activities and could be more effective than supplements with complex plant formulation in alleviating menopausal symptoms and treating osteoporosis. The transgenic yeast assays proved to be a powerful tool for assessing the in vitro estrogenic and antiestrogenic activities of dietary supplements.