Efficient protein synthesis has become a critical issue in recent biotechnology and functional protemic studies. Traditional expression of protein performed in host cells such as Escherichia coli or Saccharomyces cerevisiae is generally lengthy and costly. Cell-free protein synthesis is an attractive alternative offering simplicity and fast rate of the reaction as well as the generation of functional proteins that are difficult to obtain using in vivo systems. Furthermore, the open nature of these systems makes it amenable to manipulation allowing the investigations into the mechanism of protein synthesis itself and into the inhibition of that process by interfering molecules such as antibiotics. Here we review all the main classes of cell-free protein expression system and we emphasize their potency and recent applications in biotechnology.