Novel methods of incorporating non-native amino acids and stable isotope labels into proteins using modified tRNAs present new opportunities for basic research and biotechnology that go beyond conventional site-directed mutagenesis. tRNA-mediated protein engineering relies on the development of novel tRNAs and their misacylation with custom-designed amino acids, the recognition of special codons by the tRNAs, and the efficient expression of these modified proteins. Recent progress has been made in all these areas, including the development of more effective suppresor tRNAs and higher yield translation systems, leading to a variety of novel applications.