Cell-free protein synthesis (CFPS) represents a versatile and powerful platform for in vitro protein production, utilizing cell extracts or purified components containing cellular machinery essential for transcription and translation. Unlike traditional in vivo systems, CFPS operates independently of cellular viability and metabolic regulation, offering enhanced control over reaction conditions, reduced complexity, and faster synthesis and recovery of target proteins. Over the past few decades, substantial advancements in extract preparation, reaction optimization, and energy regeneration systems have significantly improved the efficiency, yield, and scalability of CFPS systems. These improvements have positioned CFPS as a key technology in a wide range of applications from synthetic biology and systems biology to diagnostics, high-throughput screening, biomanufacturing, and even educational settings. Its open and modular nature makes it particularly attractive for the rapid prototyping of genetic circuits, toxic or membrane protein expression, and on-demand protein synthesis. The growing availability of CFPS systems derived from various sources (including Escherichia coli, wheat germ, human cell extracts, and fully reconstituted systems) has further broadened its accessibility and applicability across disciplines. This chapter highlights the key innovations driving modern CFPS platforms, offers a critical comparison between cell-free and traditional cellular expression systems, and discusses current challenges. It also evaluates the relative strengths of different CFPS systems and explores their role in both academic research and industrial biotechnology.