Analysis of mitochondrial dysfunction is of particular importance in drug development, as it has been implicated in many common diseases and therapeutic treatments. Here, the markers of mitochondrial function and toxicity are reviewed, as well as current methods of assessment, with particular emphasis on oxygen respirometry. Fluorescence-based Respirometric Screening Technology (RST) allows convenient high-throughput analysis of oxygen consumption by cells, isolated mitochondria, enzymes, tissues and organisms, and is, therefore, of high value for such studies and general absorption, distribution, metabolism and excretion and toxicology studies. Various RST assay formats are described and specific applications are discussed. Consideration is also given to the future potential of this analytical approach.