A new bioluminescent method for coenzyme A (CoA) quantification is described. It is based on the enzymatic conversion of dehydroluciferyl-adenylate (L-AMP) into dehydroluciferyl-coenzyme A (L-CoA) by firefly luciferase (E.C. 1.13.12.7) (LUC), which causes a flash of light that can be measured in a luminometer. The method was subjected to optimization using experimental design methodologies to obtain optimum values for the concentrations of L-AMP ([L-AMP]), luciferase ([LUC]), ATP ([ATP]) and luciferin ([LH(2)]). This method has a linear response over the range of 0.25-4 microM of CoA, with a limit of detection (LOD) of 0.24 microM and a limit of quantification (LOQ) of 0.80 microM. The assay has a relative standard deviation of about 7%. By coupling this optimized procedure to bioluminescent detection, a sensible and robust method can be obtained for the analysis of CoA.