Abstract: Strong M-H⁺ ion peaks were observed for the cinnamide derivatives with tertiary amino groups in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), while no M-H⁺ ion peaks were observed on electrospray ionization mass spectrometry (ESI-MS). To investigate the possible mechanism of the M-H+ formation for these compounds, a series of experiments were performed on the effects of different matrices. In all the experiments, M-H⁺ ion peaks were observed significantly. It was considered that those cinnamide derivatives with tertiary amino groups could firstly form M⁺. or M+H⁺ ions in MALDI source with the loss of an electron or the acceptance of a proton at the tertiary amine group. Then induced by continuous collisions during the secondary ionization process, an iminium ion could be formed via a dehydrogenation process between the tertiary amino nitrogen atom and the adjacent methenyl-carbon atom, and furthermore the CN bond of the iminium ion was stabilized by forming a conjugate system with the adjacent groups. The conjugation results in the internal energy reduction of the cation, thus these ions tend to be generated abundantly in MALDI source and detected as M-H⁺ ion peaks eventually. The results of DFT calculation indicate that the dehydrogenation process to form M-H⁺ is a thermodynamic spontaneous process. Further experiments are performed with other compounds also containing the tertiary amino moieties and the results are in good agreement with above mechanism of the M-H⁺ formation.