A method is described for the preparation of CoA thioesters of fatty acids. Acyl-CoA thioester was synthesized by way of 1-acylimidazole and purified by high performance liquid chromatography. This method is applicable to the preparation of a variety of acyl-CoA thioesters of various chain lengths (from C2 to C18) and with varying degrees of unsaturation. It is particularly suitable for preparing acyl-CoA thioesters of labeled fatty acids or fatty acids available only in small amounts.