Peptide ligation is a key step in chemical protein synthesis, which allows us to produce long polypeptide chains (e.g. > 100-150 amino acids) from multiple peptide building blocks. Native chemical ligation (NCL) and its extended methods have been employed to produce not only naturally occurring proteins with/without site-specific posttranslational modifications but also artificial proteins such as mirror-image proteins and fluorophore-labelled proteins. However, chemical protein synthesis still includes issues to be solved such as the low-yield in large and hydrophobic protein synthesis and the time-consuming procedures. To upgrade and streamline the procedure of chemical protein synthesis, many sophisticated chemistries and strategies such as efficient peptide thioester synthesis, desulfurization, solubilizing tags, and one-pot multi-segment ligation, have been developed so far. In this presentation, as a novel strategy of one-pot multiple peptide ligation, “direction-switching one-pot ligation” is showcased by highlighting an orthogonal pair of protected Cys residues, thiazolidine as N-terminal Cys precursor and N-(2,2,2-trifluoroethyl) allyloxycarbonylaminomethyl (TfeAllocam)-protected Cys for C-terminal thioester precursor. In addition, production of mirror-image binder by combining mRNA display technology with chemical protein synthesis will also be presented.