This unit contains several protocols to determine the energy utilization of T cells in real‐time using a Seahorse Extracellular Flux Analyzer (http://www.seahorsebio.com). The advantages to using this machine over traditional metabolic assays include the simultaneous measurement of glycolysis and mitochondrial respiration, in real‐time, on relatively small numbers of cells, without any radioactivity. The Basic Protocol describes a standard mitochondrial stress test on the XF^e96, which yields information about oxidative phosphorylation and glycolysis, two energy‐generating pathways. The alternate protocols provide examples of adaptations to the Basic Protocol, including adjustments for the use of the XF^e24. A protocol for real‐time bioenergetic responses to T cell activation allows for the analysis of immediate metabolic changes after T cell receptor stimulation. Specific substrate utilization can be determined by the use of differential assay media, or the injection of drugs that specifically affect certain metabolic processes. Accurate cell numbers, purity, and viability are critical to obtain reliable results. © 2016 by John Wiley & Sons, Inc.