Rationale During the last decade there has been a large increase in the availability of new synthetic opioids on the European drug market. Fentanyl analogues accounted for a significant proportion of these compounds. When there is a rapid introduction of new compounds from a given chemical class onto the market, a computer‐assisted library search is not recommended and the spectra have to be interpreted individually. Therefore, the knowledge of how different groups of new psychoactive substances are fragmented can be very helpful in identifying new compounds. Methods In this study, the fragmentation patterns of 33 fentanyl derivatives were investigated using electrospray ionisation (ESI). The analyses were conducted using liquid chromatography quadrupole time‐of‐flight mass spectrometry (LC/QTOFMS). Based on measurements carried out under various conditions, the fragmentation pathways of the tested compounds that were divided into groups due to their chemical structure were established. Results The performed study allowed for the determination of characteristic ions that were formed during the fragmentation of fentanyl derivatives using ESI. Due to the high mass accuracy of the LC/ESI‐QTOFMS technique, it was proved that the cleavage of the tested molecules occurred mostly on the bonds adjacent to the nitrogen atoms. Based on the proposed fragmentation scheme, the general structure for fentanyls, and the presence of some characteristic ions, it is possible, after applying simple mathematical operations, to calculate the masses of individual substituents in the formulas of the new fentanyl analogues, which may appear on the drug market. Furthermore, based on the exact masses, it is possible to determine the formulas of these substituents. Conclusions Knowledge of the specific fragments generated under ESI conditions can be used in forensic laboratories to determine the structures of novel compounds from the group of fentanyl derivatives.