Upon activation, T cells undergo a profound reconfiguration of their metabolic profile, transitioning from a quiescent to a metabolically active state characterized by an increase in both aerobic glycolysis and mitochondrial respiration. Seahorse extracellular flux (XF) analysis is a powerful method for measuring the changes in fundamental metabolic pathways in real-time, including aerobic glycolysis and mitochondrial respiration of live T cells. This method allows a precise determination of mitochondrial performance and lactate secretion, which is measured as oxygen consumption rate (OCR) and glycolytic proton efflux rate (ECAR), respectively. By dynamically monitoring these metabolic changes, Seahorse XF analysis provides comprehensive insights into the metabolic dynamics of (activated) T cells across diverse experimental conditions or treatments.