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Hypoxia resulting from an imbalance of oxygen availability and consumption defines a metabolic cellular state with a profound impact on developmental processes, tissue maintenance, and the development of pathologies. Fluorescence imaging using genetically encoded reporters enables hypoxia and oxygen imaging with cellular resolution. Thereby unrestricted visualization of hypoxic cells and regions essentially relies on the availability of oxygen-independent fluorescent proteins like UnaG, isolated from the Japanese freshwater eel. Here, we describe the application of recently developed members of a UnaG-based hypoxia reporter family to visualize oxygenation patterns by in vitro live-cell imaging and during the ex vivo analysis of intracranial xenografted tumors. Thus, the generation of stably transfected transgenic tumor cell lines, the in vitro calibration of the genetically encoded sensors, the surgical procedures for orthotopic xenografting of tumors in mice, and workflows for the respective sample preparation and microscopy are outlined., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)