Systematic development of a UHPLC method with UV detection was performed for simultaneous determination of aglycone (genistein, daidzein, biochanin A and formononetin) and glycoside (genistin, daidzin, sissotrin, ononin) forms of isoflavones. Method development was performed by the QbD approach on three different stationary phases using the DryLab software modelling package. Optimised chromatographic separation was achieved on a Zorbax RRHD Eclipse Plus C18 column with gradient elution in a 2.0 min run time. The resolutions between eight isoflavones were found to be greater than 2.0. The developed method was validated with respect to selectivity, LOD, LOQ, linearity, precision, accuracy, stability and robustness. The proposed method was successfully applied for the analysis of isoflavones present in dietary supplements also containing various other substances. Due to the complex matrices present in the dietary supplements, a 2D-UHPLC-MS method was employed for identification and selectivity confirmation of the developed method. Analysis of investigated dietary supplements revealed deviations between the measured and labelled content of isoflavones as well as high batch to batch variations. The obtained results emphasize the need for quality control of phytoestrogen dietary supplements to provide efficient and safe products.