Mitochondria play pivotal roles in cellular energy metabolism. Most of the intracellular adenosine triphosphate (ATP) is generated by mitochondrial respiration. The Cell Mito Stress Test is a common method to measure the key parameters of mitochondrial respiration. Here, we use the HEBG2 cell line as an example to present the procedures to quantify the oxygen consumption rate using a Seahorse XFe96 extracellular flux analyzer. The optimal FCCP concentration is defined as the concentration at which maximal stimulation of OCR has been observed. The lowest concentration of FCCP that produces maximal stimulation of OCR The OCR should be conserved over 3 measurement periods such that there is neither a decrease nor an increase in OCR. In our data, 16,000 cell density and FCCP 1µM is the ideal candidate for future experiments to determine the toxicity because that produces maximal stimulation of OCR that is stable.