Mammalian cells generate ATP by mitochondrial (oxidative phosphorylation) and non-mitochondrial (glycolysis) metabolism. Cancer cells are known to reprogram their metabolism using different strategies to meet energetic and anabolic needs ( Koppenol , 2011 ; Zheng, 2012). Additionally, each cancer tissue has its own individual metabolic features. Mitochondria not only play a key role in energy metabolism but also in cell cycle regulation of cells. Therefore, mitochondria have emerged as a potential target for anticancer therapy since they are structurally and functionally different from their non-cancerous counterparts (D'Souza , 2011). We detail a protocol for measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurements in living cells, utilizing the Seahorse XF24 Extracellular Flux Analyzer (Figure 1). The Seahorse XF24 Extracellular Flux Analyzer continuously measures oxygen concentration and proton flux in the cell supernatant over time ( Wu , 2007 ). These measurements are converted in OCR and ECAR values and enable a direct quantification of mitochondrial respiration and glycolysis. With this protocol, we sought to assess basal mitochondrial function and mitochondrial stress of three different cancer cell lines in response to the cytotoxic test lead compound mensacarcin in order to investigate its mechanism of action. Cells were plated in XF24 cell culture plates and maintained for 24 h. Prior to analysis, the culture media was replaced with unbuffered DMEM pH 7.4 and cells were then allowed to equilibrate in a non-CO incubator immediately before metabolic flux analysis using the Seahorse XF to allow for precise measurements of Milli-pH unit changes. OCR and ECAR were measured under basal conditions and after injection of compounds through drug injection ports. With the described protocol we assess the basic energy metabolism profiles of the three cell lines as well as key parameters of mitochondrial function in response to our test compound and by sequential addition of mitochondria perturbing agents oligomycin, FCCP and rotenone/antimycin A. Figure 1.Overview of seahorse experiment.