Abstract Thioesters of coenzyme A (CoA) carrying different acyl chains (acyl-CoAs) are central intermediates of many metabolic pathways and donor molecules for protein lysine acylation. Acyl-CoA species largely differ in cellular concentrations and physico-chemical properties, rendering their analysis challenging. Here we compare several approaches to quantify cellular acyl-CoAs concentrations in normal and ischemic rat liver, using HPLC and LC-MS/MS for multi-acyl-CoA analysis, as well as NMR, fluorimetric and spectrophotometric techniques for quantification of acetyl-CoA. In particular, we describe a simple LC-MS/MS protocol that is suitable for the relative quantification of short and medium chain acyl-CoAs species. We show that ischemia induces specific changes in the short chain acyl-CoA relative concentrations, while mild ischemia (1-2 min), although reducing succinyl-CoA, has little effects on acetyl-CoA, and even increases some acyl-CoAs species upstream of the tricarboxylic acid cycle. In contrast, advanced ischemia (5-6 min) also reduces acetyl-CoA levels. Our approach provides the keys to accessing the acyl-CoA metabolome for a more in-depth analysis of metabolism, protein acylation and epigenetics.