© 2020, Springer Science+Business Media, LLC, part of Springer Nature. Metabolism plays an important role in the activation and effector functions of macrophages. Intracellular pathogens, such as Mycobacterium tuberculosis, subvert the immune functions of macrophages to establish an infection by modulating the metabolism of the macrophage. Here, we describe how the Seahorse Extracellular Flux Analyzer (XF) from Agilent Technologies can be used to study the changes in the bioenergetic metabolism of the macrophages induced by infection with mycobacteria. The XF simultaneously measures the oxygen consumption and extracellular acidification of the macrophages noninvasively in real time, and together with the addition of metabolic modulators, substrates, and inhibitors enables measurements of the rates of oxidative phosphorylation, glycolysis, and ATP production.