Cells require energy in the form of ATP to function. The two main ways in which cells generate energy in mammalian cells is through glycolysis and oxidative phosphorylation (OXPHOS). Glycolysis takes place in the cytosol and involves the breakdown of glucose molecules, generating ATP and pyruvate, while OXPHOS takes place in the mitochondria and is responsible for producing the majority of ATP for the cell. A dysregulation of these cellular processes has been reported in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). In order to understand the mechanisms of the disease, it is imperative to understand how the bioenergetic pathways are altered in ME/CFS. Here we describe a method for measuring mitochondrial function and glycolytic function using the Agilent Seahorse Extracellular Flux Analyzer. We have optimized these assays for use in actively proliferating lymphoblastoid cell lines that are generated from blood cells. This assay measures oxygen consumption rate and extracellular acidification rates providing an overview of mitochondrial function and efficiency and glycolytic rate and capacity, respectively. These assays are performed on live, intact cells, and enable us to view different components and measurements of energy metabolism through the injection of different compounds that stimulate or inhibit various sections of these pathways. The below method details an optimized glycolysis and mitochondrial assay for 96-well plates with modifications noted for use in 24-well plates.