Cell-free protein synthesis is advantageous for the expression of protein complexes, since it is suitable for the co-expression of two or more components of the target protein complexes. The quantity and the quality of cell-free expressed complexes are generally better than those of protein complexes expressed in conventional cell-based systems, because various parameters, such as the stoichiometry of the component proteins, can be more precisely controlled. In this chapter, we describe techniques for the expression of protein complexes by an Escherichia coli cell-free protein synthesis system, which has been successfully utilized in crystallographic structural studies.