The purpose of this study is the establishment of the rational method for the development of FRET-based biosensors. We have developed a new method to measure inositol 1,4,5-trisphosphate (IP3) concentrations, based on the two rationally designed fluorescent molecules, chemically synthesized fluorescent ligand (FL) and a fluorescent IP3-binding protein. Binding of these fluorescent molecules generates a large FRET signal, and this FRET signal is decreased by IP3 due to the displacement of FL. We call this method the “Competitive Fluorescent Ligand Assay for IP3” (CFLA-IP3). In this study, we designed novel IP3 sensors consisting of the ligand binding domain of IP3 receptor and fluorescent ligands. Finding of peptides that serve as ligand allows us to apply CFLA to develop genetically encoded FRET sensors. To achieve this goal, we try to develop a method for the screening of ligand peptide.