Methods and kits are provided for calibrating a cell-free protein synthesis reaction for optimal activity. The method includes the steps of providing an extract competent for cell-free protein synthesis (CFPS); performing cell-free protein synthesis with the extract; measuring a first reaction end-point where in vitro protein synthesis plateaus; measuring a second reaction end-point where Energy Charge of the extract declines to a level in a range from about 0.40 to about 0.80 of Energy Charge of a control extract; and adjusting Energy Charge of the extract to a level in a range from about 0.80 to about 1.0 of the control extract.