The dissociation of benzylpyridinium “thermometer” ions is widely used to calibrate the internal energy of ions produced in mass spectrometry. The fragmentation mechanism is usually believed to yield a benzylium cation, although recent studies suggest the possibility of a rearrangement leading to the tropylium isomer, which would compromise the accuracy of energy calibrations. In this study, we used IRMPD spectroscopy to probe the dissociation pathways of the p-(tert-butyl)- benzylpyridinium ion. Our results show that the formation of both benzylium and tropylium products is feasible depending on the activation regime and on the reaction time scale. Varying the trapping delays in the hexapole gives insight into a rearrangement mechanism occurring through consecutive reactions with an isomerization from benzylium to tropylium. Our work provides experimental validations for the established calibration procedure and highlights the adequacy of IRMPD spectroscopy to qualitatively resolve gas-phase rearrangement kinetics. ; Peer reviewed