Abstract: Electrospray ionization mass spectrometry (ESI-MS) is commonly used nowadays in the structural analysis of a large variety of compounds. Successful structure elucidation based on tandem mass spectrometry (MS/MS) data is dependent on correct interpretation of the fragmentation pathways. However, MS/MS based structural elucidation has been challenging due to the widespread rearrangement reactions in the fragmentation process. Thus, mastering the migration reaction mechanism in MS/MS is a key to deeply understand the fragmentation behavior and to clarify the structure elucidation of related compounds in complex matrices. In this work, the fragmentation reactions of protonated N,N-diethylaniline compounds were researched by EESI-MS. The intramolecular reactions included proton transfer, electron transfer, and hydride transfer reaction were occurred in the gas phase dissociation of protonated N,N-diethylaniline compounds, which were explored by extractive electrospray ionization tandem mass spectrometry (EESI-MS/MS). Upon collisional activation, protonaed N,N-diethylaniline underwent dissociation reaction via cleavage of the bond to give reactive ion-neutral complex (INC) intermediate. INC was found to be ubiquitous in gas-phase ion reactions. To support the proposed mechanism, deuterium-labeled experiments and the effects of different substituents on the fragmentation reaction were prepared and evaluated. The typical characteristics and basic rules of fragmentation reaction of these compounds were summarized. The implementation of this project not only enriches the research content of gas-phase ion chemistry, but also fundamentally reduces the false positive rate of detection, providing a scientific basis for structural analysis and identification of related compounds in actual samples.