Objective To analyze the fragments of 3 isoflavones and their isoflavone aglycones of Semen Sojae Praeparatum by electron spray ion trap mass spectrometry (ESI-MS), and to study the fragmentation pathway by major fragment ions. Methods Samples were fed into the instrument in the positive and negative modes, and the fragments of the samples were yielded by multi-stage ion trap mass spectrometry (ESI-MSn). The fragment ions of isoflavones and their isoflavone aglycones were analyzed. Results In the positive mode, ion peaks m/z 417, 255, 227, 199, 137, and 119 were detected for daidzin and daidzein; m/z 447, 285, 270, and 229 for glycitin and glycitein; and m/z 433, 271, 253, 243, 215, and 153 for genistin and genistein. In negative mode, ion peaks m/z 461, 415, 253, 225, 209, and 197 were detected for daidzin and daidzein; m/z 491, 445, 283, and 268 for glycitin and glycitein; and m/z 477, 269, 268, and 225 for genistin and genistein. Conclusion In the positive mode, daidzin and daidzein are fragmented by losing -Glu and -CO and Retro Diels-Alder (RDA) reaction; glycitin and glycitein are fragmented by losing -Glu, -CO, and -CH3; genistin and genistein are fragmented by losing -Glu, -CO, -H2O and RDA reaction. In negative mode, ions fragment \[M+HCOO\]- is produced by isoflavone glucosides, and daidzin and daidzein are fragmented by losing -Glu, -CO, -2CO and -CO2; glycitin and glycitein are fragmented by losing -Glu, -H, and -CH3; and genistin and genistein are fragmented by losing -Glu, -H, and -CO2.