One of the major challenges in human therapy is to develop delivery systems that are convenient and effective for tackling problems in disease treatments. In the past 20 years, liposomes have represented promising pharmaceutical carriers for drug delivery. Due to their biophysical properties, liposomes can deliver and specifically target a large set of bioactive molecules, they can protect molecules from degradation, and their composition is easily modifiable. The use of recombinant proteoliposomes containing therapeutic membrane proteins is a recently developed technology that allows biologically active proteins to penetrate across the plasma membrane of eukaryotic cells. One of the bottlenecks in this powerful delivery system lies in the production of functional therapeutic membrane proteins mainly due to their biophysical characteristics. Membrane proteins represent about 30% of the total proteins from an organism, and play a central role in drug discovery as potential pharmaceutical targets. This chapter describes the methodology for the production of bioactive proteoliposomes containing therapeutic, proapoptotic membrane proteins synthesized with an optimized cell-free expression system. We will examine (1) the design of the expression vectors and the liposome compositions compatible with the cell-free expression system; (2) the production of membrane proteins using a cell-free expression system in combination with liposomes, to obtain in a one-step reaction functional therapeutic proteoliposomes; (3) proteoliposome purification for further use in the treatment of cancer cells; and (4) the methodology for detecting apoptosis in cells after treatment. Furthermore, this system can be easily adapted for producing "difficult to express proteins" compared with the classical overexpression (bacterial or eukaryotic) systems.