The elucidation of the translation system is not only one of the most important goal in biology but also, from biotechnological point of view, provides a clue to synthesize large amount of any desired protein from mRNA. Cell-free protein synthesis system was developed as a model to study the mechanism of translation and succeeded to provide a lot of proteins even if its property suppressed cell survival but the attempts to incorporate D-amino acids into proteins with in vitro translation system have been unsuccessful and all D-amino acid-containing peptide and proteins are obtained from posttranslational modification or from nonribosomal synthesis. Here we report that novel method that is able to detect the incorporation of D-amino acid with cell-free protein synthesis system.