Mitochondrial dysfunction is increasingly associated with disease states. These organelles, responsible for adenosine triphosphate production, have been targeted for improved function in such diseases as Parkinson's, Alzheimer's, type 2 diabetes, and sarcopenia. In addition, the importance of determining if a clinical drug candidate adversely effects mitochondria function, which could lead to overt toxicity, has been recognized. Hence, assays that measure mitochondria activity have become essential in early stage drug development. Limitations of current assays that measure mitochondria membrane potential have prohibited the high-throughput performance necessary to screen current chemical space. Here, we describe a homogeneous assay to measure mitochondria membrane potential that can utilize either adherent or suspension cell types. The assay has been miniaturized to 1,536-well plate format, and was used to perform a fully automated robotic high-throughput screen of a small molecule chemical library.