We report the establishment of the O-acyl isopeptide method-based racemization-free segment condensation reaction toward future chemical protein synthesis. Peptide segments containing C-terminal O-acyl Ser/Thr residues were successfully synthesized by use of a lower nucleophilic base cocktail for Fmoc removal, and then coupled to an amino group of a peptide-resin without side reactions or epimerization. We also succeeded in performing the segment condensation in a sequential manner and in solution phase conditions as well.