The use of expensive high-energy phosphate compounds and exogenous enzymes to power protein synthesis limits eukaryotic cell-free protein synthesis. In a Saccharomyces cerevisiae crude extract CFPS platform, we demonstrate the capacity to regenerate ATP by using glucose as a secondary energy substrate. In comparison to the CrP/CrK system, batch reactions with 16 mM glucose and 25 mM phosphate produced 3.64 0.35 g mL1 of active luciferase, resulting in a 16% increase in relative protein yield. Efficient eukaryotic CFPS platforms can be developed on the basis of our demonstration.