Cell-free protein synthesis (CFPS) is a powerful protein expression platform where protein synthesis machinery is borrowed from living organisms. Target proteins are synthesized in a reaction tube together with cell extract, amino acids, energy source, and DNA. This reaction is versatile, and dynamic optimizations of the reaction conditions can be performed. The "œopen" nature of CFPS makes it a compelling candidate for many technologies and applications. This dissertation reports new and innovative applications of CFPS including 1) enzyme encapsulation in a virus-like particle, 2) detection of endocrine disrupting chemicals in the presence of blood and urine, and 3) expression of a multi-disulfide bond therapeutic protein. Two major limitations of enzymes are their instability and recycling difficulty. To overcome these limitations, we report the first enzyme encapsulation in the CFPS by immobilizing in a virus-like particle using an RNA aptamer. This technique allows simple and fast enzyme production and encapsulation We demonstrate, for the first time, the Rapid Adaptable Portable In vitro Detection biosensor platform (RAPID) for detecting endocrine disrupting chemicals (EDCs) in human blood and urine samples. Current living cell-based assays can take a week to detect EDCs, but RAPID requires only 2 hours. It utilizes the versatile nature of CFPS for biosensor protein complex production and EDC detection. Biotherapeutic protein expression in E. coli suffers from inclusion body formation, insolubility, and mis-folding. Since CFPS is not restricted by a cell wall, dynamic optimization can take place during the protein synthesis process. We report the first expression of full-length tissue plasminogen activator (tPA) using CFPS. These research works demonstrate the powerful and versatile nature of the CFPS.