Virus-like particles (VLPs) have recently received considerable attention for vaccination, drug delivery, gene therapy and material science applications. VLP-based vaccines have repeatedly demonstrated high responses from low doses due to the strong antigenic nature of their repeated ordered surface. Examples of VLP-based vaccines include the Human Papillomavirus vaccine, Gardasil, approved by the FDA in June 2006 and the Hepatitis B vaccine. By genetically and/or chemically modifying VLPs, VLPs have been used as efficient cell-specific drug/gene delivery devices and self-assembling nanostructure templates. Also, by chemically attaching antigens to the surface of VLPs, low-dose vaccines against any antigen can potentially be developed, such as the nicotine decorated Qβ bacteriophage VLP vaccine being developed by Cytos Biotechnology. Although the number of unique VLPs discovered and their applications are rapidly growing, VLP potential is limited by the current diverse, expensive, and typically low-yielding laboratory production processes available. These production processes, when scaled, often result in structurally and compositionally inconsistent products. We present Escherichia coli-based cell free protein synthesis as a production technology to overcome many of the limitations of current VLP production processes. Using this technique the self-assembling MS2 bacteriophage coat protein VLP and the self-assembling Hepatitis B core protein VLP were produced at yields 10 and 2 times greater than the best published production yields. These VLPs were found to have identical characteristics as those produced in vivo. The scalability of this technology was demonstrated without loss in production yields. To our knowledge, this is the first time a prokaryote-based in vitro transcription/translation system has generated a virus-like particle.