The fragmentation pathways in electron ionization (EI) mass spectra of a series of new N(5)-oxides of alloxazines and iso-alloxazine are presented, and compared with those of substituted alloxazines and iso-alloxazine. The EI mass spectra of these compounds showed characteristic fragmentation pathways A, B and C, started by the ejection of atomic oxygen, a HNCO molecule and an OH(*) radical, respectively. On the basis of B/E and B(2)/E spectra, the mechanism of elimination of the OH(*) radical is discussed. The influence of the methyl substituent in the benzene ring of alloxazine on the mass fragmentation pathways is described.