Tumor hypoxia or a reduction of the tissue oxygen tension is a key microenvironmental factor for tumor progression and treatment resistance in solid tumors. Because hypoxic tumor cells have been demonstrated to be more resistant to ionizing radiation, hypoxia has been a focus of laboratory and clinical research in radiation therapy for many decades. It is believed that proper detection of hypoxic regions would guide treatment options and ultimately improve tumor response. To date, most clinical efforts in targeting tumor hypoxia have yielded equivocal results due to the lack of appropriate patient selection. However, with improved understanding of the molecular pathways regulated by hypoxia and the discovery of novel hypoxia markers, the prospect of targeting hypoxia has become more tangible. This chapter will focus on the development of clinical biomarkers for hypoxia targeting.