In recent years, consumption of herbal supplements as an alternative to pharmaceutical drug therapy has increased. For example, with the health claims labeling which describes the link between soy-protein and a reduced risk of coronary heart disease (CHD), the consumption of soy and soy-derived phytoestrogens has increased dramatically. That being said, the oral bioavailability of only a few soy phytoestrogens such as Daidzein and Genestein have been previously estimated. In this paper, we present the calculated percent of rat oral bioavailability of five soy-derived phytoestrogens (Genistein, Daidzein, Biochanin A, Coumestrol, and Zearalenone) in male Sprague-Dawley rats. The plasma quantitation required for the bioavailability calculation is performed by using a rapid on-line plasma extraction procedure for the quantitative analysis. To further speed up the analysis the rats were dosed using the 'n-in-one' (cassette) protocol. The rapid on-line extraction/quantitation methodology coupled to the cassette dosing analysis of phytoestrogens is the key point of this paper. The limit of quantitation (LOQ) for each compound was 1-1000 ng/ml with each plasma sample analysis taking less than 2 min. In general the percent oral bioavailability was determined to be between 11 and 28%.