Abstract: The reaction sequence of matrix-assisted laser desorption/ionization (MALDI) was examined using various analytical techniques, including mass spectrometry and optical spectroscopy. Photoelectron emission was found to be occurred with laser fluences much less than the threshold fluence for ion production. Photoionization is the most probable initial ionization reaction in MALDI, and the photoelectrons are mainly produced from crystalline matrix because the ionization potential of matrix molecules reduced considerably in large matrix clusters. Ab initio calculations predicted that the photoionization can be achieved by using two photons of commonly used laser wavelengths. For 2,5-dihydroxybenzoic acid (DHB) and sinapinic acid (SA), the threshold fluences for photoelectron emission are unable to increase the surface temperature for material desorption. Negative ions may be produced via electron-capture ionization of matrix molecules when the laser fluence is high enough to promote material desorption. Proton and electron disproportionations may contribute to the ion production when the laser fluences further increase. Because the abundances of photoelectrons in the ion source region with laser fluences for ion production of various matrices are different, individual matrix molecule may develop a unique reaction pathway. Based on the results, a qualitative reaction sequence of MALDI is discussed.