The use of phytoestrogens has increased remarkably due to their biological activity and potential beneficial effects. An increasing number of dietary supplements containing different phytoestrogens are widely present in the market targeting postmenopausal women as a particular risk group. The content of isoflavones in dietary supplements not only differ quantitatively concerning their total isoflavone content, but also qualitatively concerning their isoflavone spectrum. The aim of the present study was to obtain data on pharmacokinetic properties for a set of 15 structurally related phytoestrogens (biochanin A, daidzein, formononetin, genistein, genistein-4,7-dimethylether, prunetin, 3,4,7-trihydroxyisoflavon, 4,6,7- trihydroxyisoflavon, 4,6,7-trimethoxyisoflavon, daidzin, genistin, ononin, sissotrin, coumestrol, coumestrol dimethylether) for the purpose of better understanding their biological activity and effects. Thereafter, based on the chromatographically determined lipophilicity, a novel, fast, selective and sensitive analytical method for simultaneous quantification of the 8 most important isoflavones in the aglycone and glycoside forms was developed. Lipophilicity of structurally related phytoestrogens was measured using reversed phase ultra-high performance liquid chromatography (RP-UHPLC). The phospholipid binding measurements were carried out using high performance liquid chromatography (HPLC) on a column with a stationary phase that contains phosphatidylcholine. The protein binding measurements were carried out using biomimetic chromatography. Caco-2 cells were used to estimate the intestinal permeability and the way of transport across the membrane of phytoestrogens test compounds. Obtained results differentiate phytoestrogens according to their structure where aglycones show significantly higher lipophilicity, IAM partitioning, AGP binding and Caco- 2 permeability comparing to glucosides. However, HSA binding was very high for all investigated compounds. Experimentally obtained pharmacokinetic parameters were compared with in silico predicted data by different computer platforms. A new RP-UHPLC method was developed for simultaneous determination of the most significant phytoestrogens from isoflavone family in aglycone (genistein, daidzein, biochanin A and formononetin) and glycoside (genistin, daidzin, sissotrin, ononin) forms without prior time-consuming process of hydrolysis. The method was validated as per ICH recommendations and was successfully applied to the analysis of various dietary supplements. Additionally to identification using external standard, the identity of selected compounds in samples was confirmed by using two-dimensional ultra-high performance liquid chromatography-tandem mass spectrometry (2D-UHPLC-MS). Analysis of dietary supplements containing phytoestrogens intended for use in postmenopausal women, using newly developed method, revealed significant deviations between measured and labelled content of investigated isoflavones.