Intracellular functional peptides with important roles in cells have been widely studied as regulators of cellular activity. Previously, we proposed a screening system for identifying intracellular functional peptides, which can be utilized to synthesize various cell-penetrating peptide (CPP)-functional peptide complexes, and the intracellular functions of the peptides can then be determined using various cell based assays. We demonstrated that the activity of a cell death-inducing peptide was increased by an amino acid substitution. However, covalent conjugation of CPPs with functional peptides may disrupt the biological characteristics of the functional peptides. In the present study, we developed a novel screening system for intracellular functional peptides combined with a dissociation system after delivery inside cells to eliminate the influence of CPPs. To construct this system, we developed a method for synthesizing CPP-functional peptide hetero-dimers containing a disulfide bond that is cleaved by the intracellular reducing environment. The system used Fmoc-Lys(ivDde)-OH to synthesize CPP and functional peptides in one molecule to selectively form a disulfide bond of hetero-dimeric peptides. Furthermore, we evaluated a single amino acid substitution library of the cell death inducing peptide (WELVVLGKL). We obtained 6 peptides with higher activity than the original peptide. Our results suggest that our new screening system can be eliminate the influence of CPPs and is useful for peptide screening inside cells. (C) 2019, The Society for Biotechnology, Japan. All rights reserved.