Adenine nucleotides (AXPs, also referred to as adenosines or adenylates) are a group of organic molecules including adenosine 5'- mono-, di-, and tri-phosphate (AMP, ADP, and ATP, respectively) that, combined, resembles an electrochemical storage cell to facilitate cellular energy storage and transfer. ATP, generated from ADP by photosynthesis, anaerobic respiration, and oxidative phosphorylation, powers many energy-requiring processes in the cell through hydrolysis of its terminal (γ) phosphate, whereas ADP is equilibrated with AMP and ATP by the adenylate kinase reaction. AXPs are major signaling molecules that regulate a wide range of anabolic and catabolic enzymes including AMP-activated protein kinase (AMPK), phosphofructokinase, and pyruvate dehydrogenase.Methods to determine concentrations of AXPs from cells and biological samples have historically relied on high-performance liquid chromatography (HPLC)/capillary electrophoresis techniques to measure [ATP] and [ADP]. However, due to its low basal concentrations, these techniques lack sufficient sensitivity to directly measure [AMP], which must be extrapolated using assumptions of adenylate kinase equilibrium that neglect AMP degradation and synthesis pathways. Here, we describe a detailed protocol to accurately measure [AXP] from cells by liquid chromatography-coupled mass spectrometry (LC/MS), applicable to a wide range of fields including our specific interest in AMPK-dependent metabolic regulation.