Objective: Trifolium pretense considered one of the most important medicinal plant which belongs to Fabaceae family, this plant commonly known as Red clover, its native region is Europe, Western Asia, and northwest Africa, but planted and naturalised in many other regions. The main compoundes found in Trifolium pretense are isoflavones which is determinrd by genistein, daidzein, biochanin A and formononetin. These compounds structurally related to estradiol-17 beta. Mangement of cancer is the main medicinal uses of Trifolium pretense in addition to decrease cholesterol and LDL and had anti- inflammatory activity, antioxidant and its effect on the Skin, Appendages, and Mucosal Status in Postmenopausal Women. This study was designed to isolate the main isoflavones from Trifolium pretense. Methods: The plant was collecting and washed by tap water to remove dirt and dust and rinsed with distilled water then dried at room temperature. Then, the plant was extracted by ethanol in a soxhlet apparatus and the extract partitioned with ethyl acetate by separatory funnel. The ethyl acetate layer was subjected to standard methods for active constituents identification, and purified by column chromatography. The collected fractions were analysed by TLC comparing with standard isoflavones (genistein, daidzein, biochanin A and formononetin). Preparative TLC was performed using 0.5 mm thickness of silica gel; the isolated bands were conducted with AR grade acetone. Iisolated isoflavones were characterized by 1H and 13-CNMR. Results: Isolated isoflavones (genistein, daidzein, biochanin A and formononetin) were identified by melting points which were identical with to that which were reported in the literature. Moreover, the ¹H-NMR and 13C-NMR analysis were used to identify the target compounds in CDCl3 solvent, and the values of chemical shifts have been discussed according to the literature of analogous compounds.