Membrane proteins are functionally and structurally diverse, making them a major group of substances that are required in ever-increasing quality and quantity. However, membrane proteins are also a class of proteins that are particularly difficult to produce in living cells. Toxic effects, protein misfolding and the formation of insoluble protein aggregates often make cell-based membrane protein synthesis impossible. For the first time, cell-free systems offer the possibility of expressing membrane proteins through time-saving, highly efficient synthesis methods in order to immediately integrate these into chip-based functional analysis. The development of a fast PCR-based method for creating matrices for the cloning-free membrane protein synthesis in prokaryotic and eukaryotic cell-free systems, as well as the development of specific vectors for efficient cell-free protein synthesis in insect cell lysates and CHO-based systems provide the basis for synthesizing complex membrane proteins. Eukaryotic protein synthesis systems allow multimeric and post-translationally modified membrane proteins to be synthesized. Modifications, such as glycosylation and signal peptide cleavage, as well as lipid modification and disulfide bridges result in an increased soluble and functional protein yield. In addition to this, newly synthesized membrane proteins can be integrated in cell-free eukaryotic systems in vesicles, making these membrane proteins available for subsequent analysis.