The ability to incorporate unnatural amino acids into biologically synthesized proteins will greatly extend the impact of protein engineering on polymer materials science. The present report describes the use of a rapid cell-free assay to assess the incorporation potential of unnatural amino acids in Escherichia coil. The assay features a coupled transcription-translation system (’Zubay system’) to screen incorporation of amino acid analogs into plasmid-encoded proteins. Activity estimates are based on the ability of an analog to compete with a radiolabeled natural amino acid, and toxicity effects are screened by monitoring incorporation of a second, unrelated amino acid. The assay was established with analogs known to be active in vivo, using a common bacterial expression vector as template DNA. Positive results were obtained with the leucine analog 5,5,5-trifluoroleucine, the proline analogs azetidine-2-carboxylic acid and thiazolidine-4-carboxylic acid, and three isomers of mono-fluorophenylalanine (o,m,p). No activity was observed for the phenylalanine analogs 2-thienylalanine and 3-thienylalanine. The results suggest that the cell-free assay will be a useful predictor of in. vivo incorporation and a useful tool in the design and synthesis of genetically engineered materials.