The main goal of this study was to establish callus cell suspension cultures of Medicago sativa L. 'Elci' and natural tetraploid Trifolium pratense L. and to compare the isoflavone production of the cultures to the original plants. The callus culture was transferred to liquid Murashige and Skoog media (MS3 and MS5) in order to establish the cell suspension cultures. The extracts were then analyzed by liquid chromatography-mass spectroscopy (LC-MS) for their isoflavones (phytoestrogen), mainly formononetin, biochanin A, daidzein, and genistein. The production of daidzein and formononetin was higher in cell suspension culture than in callus and herba of M. sativa L. 'Elci,' while biochanin A and genistein content could not be detected. On the other hand, the production of phytoestrogens was more successful in the herba of T. pratense L. than in both of the cultures. It might be suggested that T. pratense L. can be grown in larger fields, whereas M. sativa L. can be utilized to establish in vitro cultures in order to produce isoflavone compounds for pharmaceutical purposes.