Replacement of canonical amino acids with unnatural amino acids (UAAs) can provide proteins with novel physicochemical properties and biological functions. In this study, as an alternative option to conventional cell-based methods, we used a cell-free protein synthesis system as a flexible platform for facile and efficient production of UAA-containing proteins. We designed a cell-free protein synthesis system derived from the extract of Escherichia coli cells to maximize the selective incorporation of UAAs into the protein structure. First, for the purpose of avoiding competitive incorporation of canonical amino acids and UAAs, the cell extract was extensively washed using a diafiltration process to remove residual amino acids, thereby making the protein synthesis reaction completely dependent upon the exogenous addition of amino acids. In addition, the relatively low affinity of UAAs for cognate aminoacyl-tRNA synthetase was kinetically overcome by increasing the concentration of UAAs to nonphysiological levels. As a result of these modifications of the cell-free synthesis systems, we were able to produce UAAcontaining proteins at comparable yields to those of proteins made of canonical amino acids.