Abstract: The addition reactions between the common matrix α-cyano-4-hydroxy cinnamic acid (HCCA) in matrix assisted laser desorption (MALDI) and the sulfhydryl-contained compounds were investigated using matrix assisted laser desorption time of flight mass spectrometry (MALDI-TOF MS) and electrospray mass spectrometry (ESI-MS), and the corresponding reactivity was also compared in the acid, weak acid and alkaline conditions, respectively. The results showed that the sulfhydryl-contained compounds could much easily undergo addition reaction with HCCA under alkaline conditions, resulting in a mass shift of 189.0 u in the mass spectrum. In addition, the identity of the reaction product was supported by high resolution mass spectrometry analysis, and was further identified as covalent bonding products by tandem MS analysis. This work provides a proof for the addition reaction between MALDI matrix and sulfhydryl-contained compounds, so as to reduce the interference of the matrix in the MALDI-TOF MS analysis of the relevant compounds, and to provide a reference for the selection of matrix.