Antibodies that target intracellular proteins hold great promise in the development of novel therapeutic interventions for various diseases. In particular, antibodies that can cross cellular membranes have potential applications in controlling disease-related intracellular protein-protein interactions. Given the large number of cytosolic proteins and complicated interactions that are potentially involved in disease development, discovery of antibodies targeting intracellular proteins requires iterative cycles of expression and assessment of candidate antibodies. Because current cell-based expression methods do not provide sufficient throughput for production and assay of cytosol-penetrating antibodies, we integrated a cell-free protein synthesis system designed to provide optimal conditions for production of functional antibodies with a cytosol-penetration assay. The proposed approach of consolidating cell-free synthesis and cell-based assay will substantially expand the capability of discovering and engineering antibodies that can cross the cell membrane and effectively control protein-mediated cellular functions. Biotechnol. Bioeng. 2016;113: 2107-2112. © 2016 Wiley Periodicals, Inc.