The article reviews methods for assessing mitochondrial dysfunction in isolated mitochondria, intact cells, and in vivo. It emphasizes the importance of defining the specific dysfunction under investigation, such as ATP production or other functions like reactive oxygen species generation. The review discusses various techniques for measuring mitochondrial function, including fluxes, membrane potential, and respiratory control. Key methods for isolated mitochondria include mitochondrial respiratory control, which measures the increase in respiration rate in response to ADP. For intact cells, the equivalent measurement is cell respiratory control, which reports multiple aspects of ATP production and mitochondrial function. The article also highlights the advantages and disadvantages of working with isolated mitochondria versus intact cells, noting that the latter offers greater physiological relevance but is more complex. The review concludes by discussing the modular kinetic analysis of the proton circuit, which helps identify primary and secondary causes of dysfunction, and the importance of considering normalization methods when interpreting results.The article reviews methods for assessing mitochondrial dysfunction in isolated mitochondria, intact cells, and in vivo. It emphasizes the importance of defining the specific dysfunction under investigation, such as ATP production or other functions like reactive oxygen species generation. The review discusses various techniques for measuring mitochondrial function, including fluxes, membrane potential, and respiratory control. Key methods for isolated mitochondria include mitochondrial respiratory control, which measures the increase in respiration rate in response to ADP. For intact cells, the equivalent measurement is cell respiratory control, which reports multiple aspects of ATP production and mitochondrial function. The article also highlights the advantages and disadvantages of working with isolated mitochondria versus intact cells, noting that the latter offers greater physiological relevance but is more complex. The review concludes by discussing the modular kinetic analysis of the proton circuit, which helps identify primary and secondary causes of dysfunction, and the importance of considering normalization methods when interpreting results.