Saturation mutagenesis is a useful technique for the structural and functional analyses of proteins and for protein engineering. However, the extensive mutagenesis of genes and expression of mutated proteins are tedious and time-consuming. We have developed a simple and rapid method for the expression of mutated proteins with comprehensive single amino acid substitutions from single mutated genes having a four-base codon in a cell-free translation system. Twenty types of tRNA that were aminoacylated with one of the 20 proteinogenic amino acids and that contained a four-base anticodon were prepared by chemical aminoacylation. In the presence of one of the aminoacyl-tRNAs, a streptavidin mRNA with a four-base codon at the Tyr83 position was expressed in an Escherichia coli cell-free translation system. The N-terminus of the expressed proteins was fluorescently labeled using a fluorescent-labeled initiator Met-tRNA. Fluorescence imaging of an SDS-PAGE gel showed that all the amino acids are incorporated in response to the four-base codon; however, the incorporation efficiency was dependent on the structure of the side chains. Streptavidin mutants with comprehensive amino acid substitutions at the Tyr83, Arg84, and Tyr54 positions were used for analyzing their biotin-binding activity by dot blot analysis. These results demonstrate that this method is effective for the expression and analysis of mutated proteins with comprehensive amino acid substitutions at desired positions.