Acetyl phosphate (acetyl-P) serves critical roles in coenzyme A recycling and ATP synthesis. It is the intermediate of the Pta-AckA pathway that inter-converts acetyl-coenzyme A and acetate. Acetyl-P also can act as a global signal by donating its phosphoryl group to specific two-component response regulators. This ability derives from its capacity to store energy in the form of a high-energy phosphate bond. This bond, while critical to its function, also destabilizes acetyl-P in cell extracts. This lability has greatly complicated biochemical analysis, leading in part to widely varying acetyl-P measurements. We therefore developed an optimized protocol based on two-dimensional thin layer chromatography that includes metabolic labeling under aerated conditions and careful examination of the integrity of acetyl-P within extracts. This protocol results in greatly improved reproducibility, and thus permits precise measurements of the intracellular concentration of acetyl-P, as well as that of other small phosphorylated molecules.