In the field of chemical biology, methods for controlling peptidyl function by a stimulus are attracting increasing attention. Recently, we reported a stimulus-responsive peptide which can be cleaved after exposure to a stimulus. In this study, we developed a FRET-based assay system to estimate the kinetics of the stimulus-induced processing (peptide bond cleavage) reaction. Based on the FRET system, it was clarified that introduction of a sterically less-hindered or polar residue at the position adjacent to the stimulus-responsive amino acid accelerates the processing reaction