High-performance liquid chromatography-UV-electrospray ionization-mass spectrometric detector (HPLC-UV-ESI-MSD) method for determination of isoflavones in red clover (Trifolium pratense L.) and related species has been developed. The separated isoflavones including aglycones, glycosides and glycoside malonates, were individually analyzed and identified by their molecular ions and characteristic fragment ion peaks using LC-MSD under MS and MS-MS mode, and in comparison with the standard isoflavones. A total of 31 isoflavones were detected in red clover. Several isoflavones were also identified for the first time in related species, T. repense L. (white clover), T. hybridum L. (alsike clover) and T. campestre Schreber (hop trefoil). Based on reversed phase HPLC, all 10 isoflavone aglycones, daidzein, formononetin, genistein, pseudobaptigenin, glycitein, calycosin, prunetin, biochanin A, irilone and pratensein in acidic hydrolyzed extracts were successfully separated within 40 min and quantified individually by UV and MS detectors. For the 10 target compounds, the investigated concentrations ranged from approximately 24 to approximately 12500 ng/ml for UV detection and approximately 6 to approximately 3125 ng/ml for MS detection, and good linearities (r2 > 0.999 for UV and r2 > 0.99 for MS) for standard curves were achieved for each isoflavone. The accuracy and repeatability (n = 10) were within 15% for these 10 compounds. This is the first method reported that enables the simultaneous quantitation of all 10 isoflavone aglycones in red clover and related species.