This study has elucidated the fragmentation pathway for deprotonated isoflavones in electrospray ionization using MSn ion trap mass spectrometry and triple quadrupole mass spectrometry. Genistein-d4 and daidzein-d3 were used as references for the clarification of fragment structures. To confirm the relationship between precursor and product ions, some fragments were traced from MS2 to MS5. The previous literature for the structurally related flavones and flavanones located the loss of ketene (C2H2O) to ring C, whereas the present fragmentation study for isoflavones has shown that the loss of ketene occurs elsewhere at ring A. In the further fragmentation of the [M-H-CH3]•- radical anion of methoxylated isoflavones, loss of a hydrogen atom was commonly found. [M-H-CH3COB-ring]- could be a characteristic fragment ion of glycitein and be used to differentiate glycitein from its other isomers. Neutral losses of CO and CO2 were prominent in the fragmentation of deprotonated anions in the ion trap mass spectrometry, whereas recyclization cleavage accounted for a very small proportion. In comparison with triple quadrupole mass spectrometry the use of the ion trap MSn mass spectrometry has the advantage of better elucidation of the relationship between precursor and product ions.