We developed and validated three different sample preparation and extraction methods followed by HPLC-MS/MS (negative electrospray ionization) analysis for the quantification of estrogenic isoflavones (formononetin, daidzein, equol, biochanin A, and genistein) and coumestrol in red clover, soil, and manure. Plant and manure samples were solid-liquid extracted, whereas soil was extracted with accelerated solvent extraction. Absolute recoveries were between 80 and 93%, 20 and 30%, and 14 and 91% for plant, soil, and manure samples, respectively. Relative recoveries ranged from 75 to 105% for all matrices, indicating that isotope-labeled internal standards (¹³C₃-formononetin, ¹³C₃-daidzein, ¹³C₃-equol, ¹³C₃-biochanin A, and ¹³C₃-genistein) were capable to compensate for losses during analysis. The limits of detection in red clover, soil, and manure were 3-9 μg/g(dryweight(dw)), 0.6-8.2 ng/g(dw), and 34.2 ng/g(dw) to 17.0 μg/g(dw), respectively. Formononetin was the most dominant compound in red clover plants (up to 12.5 mg/g(dw)) and soil (up to 3.3 μg/g(dw)), whereas equol prevailed in manure (up to 387 μg/g(dw)).