With the rapid development of cell-free biotechnology, more and more cell-free protein synthesis (CFPS) systems have been established and optimized for protein expression in vitro. Here, we aim to improve the productivity of a newly developed Streptomyces-based CFPS system. Protein translation in CFPS systems depends on the entire endogenous translation system from cell lysates. However, lysates might lack such translation-related elements, limiting the efficiency of protein translation and therefore the productivity of CFPS systems. To address this limitation, we sought to add protein translation related factors to CFPS reactions. By doing this, the protein yield of EGFP was significantly improved up to approximately 400 μg/mL. In this chapter, we mainly describe the preparation of Streptomyces cell extracts, expression and purification of nine translation related factors, and optimization of the Streptomyces-based CFPS system for enhanced protein expression.