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Isoflavone is one of the phytoestrogens that have estrogenic effects, so it is usually served as an active ingredient for quality control of traditional Chinese medicines rich in isoflavones. Nine isoflavones commonly found in traditional Chinese medicines were separated in 30 min using mixed micellar liquid chromatography. The mobile phase consisted of 0.08 M sodium dodecylsulfate and 6.05 mM β‐cyclodextrin:methanol (87:13, v/v) at pH 3 and eluted isocratically at 1 mL/min through a C18 column. In this study, we systematically optimized the chromatographic conditions including the pH, the composition and concentration of surfactants, the type and ratio of organic solvents, and column temperature. The method was validated according to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use guidelines. There is no report using micellar liquid chromatography to detect isoflavones, and the optimized method has been successfully applied to quantify isoflavones in red clover and Radix Puerariae. This method is efficient, cheap, and convenient. Finally, we verified the existence of supramolecular amphiphilic vesicles in the mobile phase by transmission electron microscopy to explain the increased chromatographic efficiency.