Hypoxia, a condition of insufficient O2 to support metabolism, occurs when the vascular supply is interrupted, as in stroke or myocardial infarction, or when a tumor outgrows its vascular supply. When otherwise healthy tissues lose their O2 supply acutely, the cells usually die, whereas when cells gradually become hypoxic, they adapt by up-regulating the production of numerous proteins that promote their survival. These proteins slow the rate of growth, switch the mitochondria to glycolysis, stimulate growth of new vasculature, inhibit apoptosis, and promote metastatic spread. The consequence of these changes is that patients with hypoxic tumors invariably experience poor outcome to treatment. This has led the molecular imaging community to develop assays for hypoxia in patients, including regional measurements from O2 electrodes placed under CT guidance, several nuclear medicine approaches with imaging agents that accumulate with an inverse relationship to O2, MRI methods that measure either oxygenation directly or lactate production as a consequence of hypoxia, and optical methods with NIR and bioluminescence. The advantages and disadvantages of these approaches are reviewed, along with the individual strategies for validating different imaging methods. Ultimately the proof of value is in the clinical performance to predict outcome, select an appropriate cohort of patients to benefit from a hypoxia-directed treatment, or plan radiation fields that result in better local control. Hypoxia imaging in support of molecular medicine has become an important success story over the last decade and provides a model and some important lessons for development of new molecular imaging probes or techniques.