Measurement of tumor hypoxia is required for the diagnosis of tumor and the evaluation of therapeutic outcome. Currently, invasive and noninvasive techniques being exploited for tumor hypoxia measurement include polarographic needle electrodes, immunohistochemical (IHC) staining, magnetic resonance imaging (MRI), radionuclide imaging (positron emission tomography [PET] and single-photon emission computed tomography [SPECT]), optical imaging (bioluminescence and fluorescence), and hypoxia imaging endoscopy. This review provides a summary of the modalities available for assessment of tissue oxygenation as well as a discussion of current arguments for and against each modality, with a particular focus on noninvasive hypoxia imaging with emerging agents and new imaging technologies intended to detect molecular events associated with tumor hypoxia.