An enzyme-coupled, colorimetric method for the assay of acyl-CoA synthetase is described. Acyl-CoA formed from fatty acid and CoA by acyl-CoA synthetase was dehydrogenated by acyl-CoA oxidase. Hydrogen peroxide produced was then converted into formaldehyde in the presence of methanol by catalase. The formaldehyde reacted with a triazole compound, 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole, in an alkaline condition to form a purple dye, and the absorbance was measured spectrophotometrically.