The role of cations in the fragmentation of polymer nitroxide labile end groups, as often deplored in matrix-assisted laser desorption/ionization, was investigated. A combination of different spectroscopic techniques and theoretical calculations was implemented to address the mechanistic aspect of the end-group cleavage in a nitroxide-terminated poly(ethylene oxide) macroinitiator agent upon activation in mass spectrometry. When occurring during the ionization process, the homolytic cleavage of a C-ON bond in the end group results in the release of a nitroxide moiety and prevents the intact oligomer from being observed. In contrast, electrospray allowed oligomer ionization in a more gentle process and was used here in conjunction with tandem mass spectrometry to identify factors influencing the release of the end group. Collision-induced dissociation of intact electrosprayed oligomers showed that the elimination of the nitroxide radical depends on the cationizing agent. This fragmentation readily occurs from alkali adducts, whereas it is hardly observed from oligomer adducted with divalent cations such as Cu2+, Zn2+, or Ca2+. Nuclear magnetic resonance experiments actually revealed that the presence of divalent cations in solution would allow the formation of carboxylic salts, inducing an internal proton transfer in the end group. Upon protonation, the C-ON bond dissociation energy increases by about 25 kcal/mol, as indicated by theoretical calculations. This suggests that the release of the nitroxide radical would no longer efficiently compete with alternative dissociation pathways, as observed in MS/MS experiments. Oil the basis of the structural details provided by NMR and calculations, dissociation mechanisms Supported by accurate mass data were proposed to account for all detected fragment ions.