Fatty acids are utilized to maintain cellular energy/adenine nucleotide balance under times of energetic stress such as during endurance exercise or fasting. It has long been recognized that fatty acids stimulate their own oxidation through a mechanism involving allosteric inhibition of acetyl-CoA carboxylase (ACC) and reductions in malonyl-CoA. We have recently described a parallel pathway by which long-chain fatty acid-CoAs bind to and activate the AMP-activated protein kinase (AMPK) at the allosteric drug and metabolic (ADaM) binding site. Increases in AMPK activity lead to the phosphorylation and inhibition of ACC which is essential for fatty acids to stimulate fatty acid oxidation. Here, we describe the methods to detect fatty acyl-CoA-induced activation of AMPK in cell-free assays, primary mouse hepatocytes, and in the liver of mice. These methodologies will be useful to allow further investigations into the importance of this fatty acid sensing axis in regulating metabolism and provide a framework for future studies investigating whether there may be other natural ligands targeting the ADaM binding site of AMPK.