A dynamic microdialysis sampling method with liquid chromatography-diode array detection and time-of-flight mass spectrometry (LC-DAD-TOF/MS) analysis was developed to investigate rat microsomal metabolisms of calycosin and formononetin, and their drug-drug interactions. Two hydroxylated metabolites from calycosin, and three hydroxylated or 4'-O-demethylated derivatives from formononetin were detected and identified after co-incubation with microsomes. Calibration curves offered linear ranges of two orders of magnitude with r(2)>0.999 for calycosin, formononetin and daidzein. The quantitative LC method provides a range of 0.028-0.034microg/mL for limits of detection, overall precision less than 5% and accuracy less than 3% by RSD. Besides, calycosin and formononetin were found to produce the depressive effect on the CYP450 enzyme reaction, and inhibit phase I enzyme reaction of each other when they are concurrent. Dynamic microdialysis sampling with LC-DAD-TOF/MS analysis developed in this work is a powerful tool for in vitro metabolism studies of drugs and metabolic interactions.