Acyl-Coenzyme A (CoA) thioesters are the principal form of activated carboxylates in cells and tissues. They are employed as acyl carriers that facilitate the transfer of acyl groups to lipids and proteins. Quantification of medium- and long-chain acyl-CoAs represents a significant bioanalytical challenge because of their instability. Stable isotope dilution liquid chromatography/selected reaction monitoring-mass spectrometry (LC/SRM-MS) provides the most specific and sensitive method for the analysis of CoA species. However, relevant heavy isotope standards are not available and they are challenging to prepare by chemical synthesis. Stable isotope labeling by essential nutrients in cell culture (SILEC), developed originally for the preparation of stable isotope labeled short-chain acyl-CoA thioester standards, has now been extended to medium-chain and long-chain acyl-CoAs and used for LC/SRM-MS analyses. Customized SILEC standards with >98% isotopic purity were prepared using mouse Hepa 1c1c7 cells cultured in pantothenic-free media fortified with [(13) C3 (15) N1 ]-pantothenic acid and selected fatty acids. A SILEC standard in combination with LC/SRM-MS was employed to quantify cellular concentrations of arachidonoyl-CoA (a representative long-chain acyl-CoA) in two human colon cancer cell lines. A panel of SILEC standards was also employed in combination LC/SRM-MS to quantify medium- and long-chain acyl-CoAs in mouse liver. This new SILEC-based method in combination with LC/SRM-MS will make it possible to rigorously quantify medium- and long-chain acyl-CoAs in cells and tissues. The method will facilitate studies of medium- and long-chain acyl-CoA dehydrogenase deficiencies as well as studies on the role of medium- and long-chain acyl-CoAs in cellular metabolism.