Today, CHO cells are the market-dominating mammalian cell line for the production of complex therapeutic proteins. Despite this outstanding potential of CHO cells, no high-yield cell-free system based on translationally active lysates from these cells has been reported so far. In this work, Brodel, Sonnabend, and Kubick address this fact by establishing a novel cell-free protein expression system based on extracts from cultured CHO cells. The developed batch-type cell-free coupled transcription-translation system enables the synthesis of a broad range of target proteins such as cytosolic proteins, secreted proteins, membrane proteins embedded into endogenous microsomes, and glycoproteins. Hence, the presented in vitro translation system is a powerful tool for the fast and convenient optimization of expression constructs, the specific labeling of integral membrane proteins and the cell-free production of posttranslationally modified proteins.