Cell-free protein synthesis (CFPS) has emerged as a powerful platform technology to help satisfy the growing demand for simple, affordable, and efficient protein production. In this article, we describe a novel CFPS platform derived from the popular bio-manufacturing organism Saccharomyces cerevisiae. By developing a streamlined crude extract preparation protocol and optimizing the CFPS reaction conditions we were able to achieve active firefly luciferase synthesis yields of 7.7 +/- 0.5 mu gmL(-1) with batch reactions lasting up to 2h. This duration of synthesis is the longest ever reported for a yeast CFPS batch reaction. Furthermore, by removing extraneous processing steps and eliminating expensive reagents from the cell-free reaction, we have increased relative product yield (mu g protein synthesized per $ reagent cost) over an alternative commonly used method up to 2000-fold from approximate to 2x10(-4) to approximate to 4x10(-1)mu g$(-1), which now puts the yeast CPFS platform on par with other eukaryotic CFPS platforms commercially available. Our results set the stage for developing a yeast CFPS platform that provides for high-yielding and cost-effective expression of a variety of protein therapeutics and protein libraries. Biotechnol. Bioeng. 2013;110: 2643-2654. (c) 2013 Wiley Periodicals, Inc.