A large amount of energy used for nutrient processing and cellular functions is essential for tumorigenesis. Total intracellular adenosine triphosphate (ATP) is mainly generated by glycolysis and mitochondrial oxidative phosphorylation. Here, we provide a protocol for measurements of energy metabolism in cancer cells by using Seahorse XF24 Extracellular Flux analyzer. Specifically, this machine measures glycolysis by analyzing the extracellular acidification rate (ECAR) and measures mitochondrial oxidative phosphorylation on the basis of the oxygen consumption rate (OCR), through real-time and live cell analysis. This protocol is provided for researchers who are unfamiliar with the method and to aid them in carrying out the technique successfully.