Acyl-CoAs are thioester compounds that have a pivotal role in various metabolic processes such as fatty acid beta-oxidation, biosynthesis of lipids, signaling, and xenobiotics metabolism . The most important biological function of acyl-CoAs is in the metabolism of fatty acids via beta-oxidation. The fatty acid beta-oxidation (FAO) process in the liver breaks down fatty acids (FA) to produce adenosine triphosphate (ATP) in low glucose conditions . Acyl-CoAs are formed when a FA forms a thioester bond with Coenzyme-A (CoA) . These acyl-CoAs are transported inside the mitochondria for the entire process of FAO. Fatty acid oxidation disorders (FAOD) occur due to the deficient activity of the enzymes or transporter proteins involved in the pathway, which results in the accumulation of acyl-CoA esters . The acyl-CoA accumulation profile provides information on the type of fatty acid oxidation disorder (FAOD). Intracellular acyl-CoA levels are important reporters of metabolic health and their accumulation in case of FAOD makes them interesting biomarkers . Apart from FAOD, these compounds are also involved in progression of cancer , diabetes , precursors for lipid synthesis and ketone bodies. Since acyl-CoA are involved in numerous physiological and pathophysiological pathways, it is important to develop analytical methods for their identification and quantification.