Ribosome display is a powerful polymerase chain reaction-based in vitro display technology that is well suited to the selection and evolution of proteins. This technology exploits cell-free translation to achieve coupling of phenotype and genotype by the production of stabilized ribosome complexes, whereby translated protein and their cognate mRNA remain attached to the ribosome. The Escherichia coli S30 extract for in vitro display of an mRNA library has proven to be very successful for the evolution of high-affinity antibodies and the optimization of defined protein characteristics. These methodologies will enable the end user to successfully perform ribosome display selections.