AbstractAdenosine monophosphate (AMP)‐activated protein kinase (AMPK) is a master regulator of cellular metabolism, phosphorylating a variety of downstream targets throughout the cell. Subcellular AMPK activity results in regulation of glycolysis, lipid and protein biosynthesis, mitochondrial function, and gene expression. But how AMPK senses and responds to stimuli in a compartment‐specific manner is not well understood, leaving an incomplete picture of compartmentalized AMPK activity. Key tools for studying subcellular AMPK activity are genetically encoded AMPK activity reporters (AMPKARs), which allow for the quantitative visualization of subcelluar AMPK activity. However, many AMPKARs suffer from poor dynamic range and sensitivity, limiting their application. I recently reported the development of a new excitation‐ratiometric (ExRai) AMPKAR, a single‐fluorophore AMPKAR with enhanced dynamic range for detection of subtle, subcellular AMPK activity. I used ExRai AMPKAR to study subcellular AMPK activity at several locations, including the lysosome and mitochondria, identifying new mechanisms for the regulation of AMPK activity. Here, I describe the use of ExRai AMPKAR to image subcellular AMPK activity in mouse embryonic fibroblasts using both widefield and confocal microscopy. I also describe the culture of mouse embryonic fibroblasts. Through the use of ExRai AMPKAR, subcellular AMPK activity can be illuminated to better understand how this central kinase regulates cellular metabolism. © 2023 The Author. Current Protocols published by Wiley Periodicals LLC.Basic Protocol: Imaging subcellular AMPK activity using ExRai AMPKARSupport Protocol 1: Culturing of mouse embryonic fibroblasts for live‐cell imagingSupport Protocol 2: Live‐cell imaging of ExRai AMPKAR using confocal microscopy