The entire complement of proteins expressed by the genome of an organism under a set of defined conditions represents the proteome. The need for rapid, efficient, and high-throughput technologies to understand the proteome of organisms have resulted in the development of protein microarrays. Traditional, cell-based technologies for the generation of protein arrays involve laborious and protracted procedures, which also pose several other technical problems. These limitations have been suitably overcome by cell-free expression systems that carry out rapid, in situ synthesis of proteins from their corresponding DNA templates. This article provides an overview of commonly used cell-free systems that have been used for the generation of protein microarrays, with special emphasis on the diverse applications of cell-free synthesis-based protein microarrays.