Oxygen is the terminal electron acceptor in the mitochondrial electron transport chain and therefore is required for the generation of energy through oxidative phosphorylation. In environments of decreased oxygen levels (hypoxia), organisms have developed an adaptive response through the activation of the hypoxia-inducible transcription factor (HIF) to maintain their energetic demand. In order to sense hypoxic environments, cells have developed oxygen-sensing machinery that allows for the activation of HIF. The mitochondrial electron transport chain is required for the oxygen-sensing pathway. This chapter outlines methods used to explore the role of the electron transport chain and a by-product of electron transport, reactive oxygen species, in oxygen sensing.