Since Nirenberg and Matthaei used cell-free protein synthesis (CFPS) to elucidate the genetic code in the early 1960s [1], the technology has been developed over the course of decades and applied to studying both fundamental and applied biology [2]. Cell-free synthetic biology integrating CFPS with synthetic biology has received attention as a powerful and rapid approach to characterize and engineer natural biological systems. The open nature of cell-free (or in vitro) biological platforms compared to in vivo systems brings an unprecedented level of control and freedom in design [3]. This versatile engineering toolkit has been used for debugging biological networks, constructing artificial cells, screening protein libraries, prototyping genetic circuits, developing biosensors, producing metabolites, and synthesizing complex proteins including antibodies, toxic proteins, membrane proteins, and novel proteins containing nonstandard (unnatural) amino acids. The Methods and Protocols "Cell-Free Synthetic Biology" Special Issue consists of a series of reviews, protocols, benchmarks, and research articles describing the current development and applications of cell-free synthetic biology in diverse areas. [...].