Isoquinoline alkaloids, which are one of the most important types of alkaloids, are extensively distributed in herbal medicines. However, systematic and comprehensive investigations of the fragmentation behaviours of isoquinoline alkaloids have rarely been reported. Therefore, the goal of the present study is to simultaneously investigate the collision-induced dissociation patterns and the corresponding mechanism of isoquinoline alkaloids by mass spectrometry (MS) combined with computations. Nineteen types of isoquinoline alkaloids (66 compounds) were used as references to identify the characteristic fragmentation behaviours by quadrupole time-of-flight mass spectrometry (Q-TOF/MS) in positive electrospray ionization (ESI) mode. These types of isoquinoline alkaloids were divided into three categories primarily by the characteristic [M-NHR1R2]+ (R1 and R2 represent the substituent groups of the N-atom) fragment ions. High- and low-abundance [M-NHR1R2]+ ions were observed respectively for type I (1-13) and type II (14-29) alkaloids, respectively; however, the characteristic fragments were not detected for type III alkaloids (30-66) because of the existence of a p-π conjugated system. Each type of alkaloid was further classified by its characteristic fragmentation patterns and fragment ions. In addition, isoquinoline alkaloid with vicinal methoxy and hydroxy, vicinal methoxy, methylenedioxy, methoxy, and quaternary N-methyl groups could form the characteristic fragments by the loss of CH3OH, CH4, CH2O or CO, CH3 and CO, and CH3 moieties, respectively. The mechanisms of some interesting fragmentation behaviours, such as the formation of [M-NH3]+ and [M-CH3]+ fragment ions, were further demonstrated by computational chemistry. These characteristic fragmentation behaviours and fragment ions of isoquinoline alkaloids provide a solid foundation for the rapid and high-efficiency structural elucidation of similar metabolites in plant-derived medicines.