In　the　literature,　it　is　reported　that　the　protonated　ketotifen　mainly　undergoes　C=C　double　bond　cleavage　in　electrospray　ionization　tandem　mass　spectrometry　(ESI-MS/MS);　however,　there　is　no　explanation　on　the　mechanism　of　this　fragmentation　reaction.　Therefore,　we　carried　out　a　combined　experimental　and　theoretical　study　on　this　interesting　fragmentation　reaction.　The　fragmentation　of　protonated　ketotifen　(m/z　310)　always　generated　a　dominant　fragment　ion　at　m/z　96　in　different　electrospray　ionization　mass　spectrometers　(ion　trap,　triple　quadrupole　and　linear　trap　quadrupole　(LTQ)-orbitrap).　The　mechanism　of　the　generation　of　this　product　ion　(m/z　96)　through　the　C=C　double　bond　cleavage　was　proposed　to　be　a　sequential　hydrogen　migration　process　(including　proton　transfer,　continuous　two-step　1,2-hydride　transfer　and　ion-neutral　complex-mediated　hydride　transfer).　This　mechanism　was　supported　by　density　functional　theory　(DFT)　calculations　and　a　deuterium　labeling　experiment.　DFT　calculations　also　showed　that　the　formation　of　the　product　ion　m/z　96　was　most　favorable　in　terms　of　energy.　This　study　provides　a　reasonable　explanation　for　the　fragmentation　of　protonated　ketotifen　in　ESI-MS/MS,　and　the　fragmentation　mechanism　is　suitable　to　explain　other　C=C　double　bond　cleavage　reactions　in　mass　spectrometry.　Copyright　(C)　2016　John　Wiley　&　Sons,　Ltd.