A simple and specific LC-DAD-ESI-MS/MS method has been developed and applied for the primary investigation of the chemical constituents absorbed or metabolized in vivo, after the rat oral administration of Erxian Decoction (EXD), a Chinese medicine prescription for menopausal syndromes. Through the online ESI-MSn analysis, a total of 35 compounds have been identified or tentatively characterized from the seven tested samples, and 13 of them were unambiguously identified through a direct comparison of the retention time, UV spectra and MSn fragmentation patterns with the authentic ones. The results showed that 21 compounds were detected from rat plasma, 20 compounds were detected from rat kidneys and adrenal glands, 19 compounds were detected from rat ovaries, 12 compounds were found in rat intestines, nine compounds were identified from rat livers and nine compounds were detected from rat brains at certain time points after oral administration of the eff ective EXD fraction. Copyright © 2009 John Wiley & Sons, Ltd.