There is accumulating evidence that mitochondrial respiratory malfunction is associated with aging-associated complex diseases. However, progress in our understanding of these diseases has been hampered by the sensitivity and throughput of systems employed to quantify dysfunction and inherent limitations of the biological systems studied. In this review, we describe and contrast two methodologies that have been developed for measuring mitochondrial function to address the need for improved sensitivity and increased throughput. We then consider the utility of each methodology in studying three biological systems: isolated mitochondria, cultured cells, and cell fibers and tissues. Finally, we discuss the application of each methodology in the study of mitochondrial dysfunction in Alzheimer's disease, type 2 diabetes mellitus, and aging-associated autophagy impairment and mitochondrial malfunction. We conclude that the methodologies are complementary, and researchers may need to examine multiple biological systems to unravel complex diseases of aging.