In this paper, an accurate and route method was developed to quantitative determine daidzein, genistein, glycitein, daidzin, glycitin, 6"-O-acetyldaidzin, 6"-O-acetylglycitin and 6"-O-acetylgenistin contents in selected high and low isoflavones in nutrition supplements by on line liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry (LC-APCI-MS). Improved extraction and hydrolysis methods of the isoflavones from three nutrition supplements were also studied and a rapid extraction method was developed. Comparison of different MS2 and MS3 spectra of isoflavones and some unknown compounds were also explored and proposed pathway fragments of nine isoflavones were first systematically suggested.