Fatty acid oxidation (FAO) is a key bioenergetic pathway often dysregulated in diseases. The current knowledge on FAO regulators in mammalian cells is limited and sometimes controversial. Previous FAO analyses involve nonphysiological culture conditions or lack adequate quantification. We herein described a convenient and quantitative assay to monitor dynamic FAO activities of mammalian cells in physiologically relevant settings. The method enabled us to assess various molecular and pharmacological modulators of the FAO pathway in established cell lines, primary cells and mice. Surprisingly, many previously proposed FAO inhibitors such as ranolazine and trimetazidine lacked FAO-interfering activity. In comparison, etomoxir at low micromolar concentrations was sufficient to saturate its target proteins and to block cellular FAO function. Oxfenicine, on the other hand, acted as a partial inhibitor of FAO. As another class of FAO inhibitors that transcriptionally repress FAO genes, antagonists of peroxisome proliferator-activated receptors (PPARs), particularly that of PPAR?, significantly decreased cellular FAO activity. Our assay also had sufficient sensitivity to monitor upregulation of FAO in response to environmental glucose depletion and other energy-demanding cues. Altogether this study provided a reliable FAO assay and a clear picture of biological properties of potential FAO modulators in the mammalian system.