Azoloazines possess a wide spectrum of biological activity. For the analysis of new drugs of the azoloazine series, reliable methods of identification and quantitative analysis are necessary. Mass spectrometry is one of the main methods for the analysis of drugs and their metabolites in a biological matrix. Information about the main fragmentation pathways of these substances favors their reliable identification. In this study, the fragmentation pathways of the protonated triazavirin molecule, which is an antiviral agent from the azoloazine series, were studied. The experiments were carried out using high resolution tandem mass spectrometry with an electrospray ionization source. The directions of fragmentation of the compound in the collision cell were confirmed by pseudo-MS3 experiments, which are possible due to dissociation in the ion source, and by comparative data analysis of triazavirin analogs labeled with stable isotopes. It was found that during dissociation in the ion source, two types of ions associated with the loss of the nitro group are formed, in contrast to fragmentation in the collision cell. The formation of basic product ions occurs due to the loss of substituents in the heterocyclic structure with the release of neutral molecules or radicals, and also as a result of reactions affecting the integrity of the triazolotriazine heterocyclic system. The information presented in this work may be also useful in the study of structurally similar compounds.