Metabolic engineering is traditionally practiced with whole cell systems. However, the engineering of metabolism in a cell-free environment is also possible (Michel-Reydellet et al., 2004). In fact, in vitro reactions have several advantages over in vivo applications such as the ability to sample easily and to control reaction conditions more precisely. Better understanding of the cellular extract used in cell-free reactions has allowed complex, interrelated metabolic processes to be activated in a single test tube outside of the cell (Jewett and Swartz, 2004a). In this chapter, we highlight some of the advantages of cell-free biology for metabolic engineering and describe improvements made to cell-free reactions by stabilizing amino acids, activating complex metabolism, and engineering cell-free extracts that produce disulde-bonded proteins.