Nitroimidazoles are important compounds in medicine, biology, and the food industry. The growing need for their structural assignment, as well as the need for the development of the detection and screening methods, provides the motivation to understand their fundamental properties and reactivity. Here, we investigated the decomposition of protonated ronidazole [Roni+H]+ in low-energy and high-energy collision-induced dissociation (CID) experiments. Quantum chemical calculations showed that the main fragmentation channels involve intramolecular proton transfer from nitroimidazole to its side chain followed by a release of NH2CO2H, which can proceed via two pathways involving transfer of H+ from (1) the N3 position via a barrier of TS2 of 0.97 eV, followed by the rupture of the C-O bond with a thermodynamic threshold of 2.40 eV; and (2) the-CH3 group via a higher barrier of 2.77 eV, but with a slightly lower thermodynamic threshold of 2.24 eV. Electrospray ionization of ronidazole using deuterated solvents showed that in low-energy CID, only pathway (1) proceeds, and in high-energy CID, both channels proceed with contributions of 81% and 19%. While both of the pathways are associated with small kinetic energy release of 10-23 meV, further release of the NO• radical has a KER value of 339 meV.