An enzymatic method was proposed for determination of acetylcarnitine (AcCar), even when carnitine (Car), non-acetylated form, co-exists. The method is consisted of four enzymatic reactions: First, AcCar is hydrolysed by acylcarnitine hydrolase to yield acetate; followed by the other three reactions coupled with three enzymes, respectively, acetate kinase, pyruvate kinase and lactate dehydrogenase; finally, the acetate formation causes a decrease in NADH. The amount of AcCar is then evaluated as the change in absorbance at 340 nm. The reagent composition of the reaction mixture was determined, and the characteristics of the method were investigated. The dilution test showed a good linearity over a wide range. The precision and accuracy tests produced satisfactory results. The co-existence of Car gave no effect on the measurement. The present method was found to be used easily, simply and rapidly for the selective determination of AcCar.