A variety of in vitro and ex-vivo cell and tissue models are being used in biomedical research, but for many of them control of the cellular microenvironment, particularly oxygenation state and intracellular O2 levels, is inadequate. Since O2 is a key parameter and biomarker of cellular function, implementation of reliable in situ control and knowledge of actual O2 levels in different compartments of biological samples is of critical importance. The versatile and flexible technology of O2 sensing and imaging based on phosphorescence quenching provides such capabilities. In recent years, various O2 sensing systems, which operate with solid-state sensors, soluble probes or imaging (nano)sensors in conjunction with portable handheld instruments, commercial plate readers or live cell imaging platforms, have been developed, which are suitable for routine use in many research labs to perform a range of important analytical and biomedical tasks. Here we overview the available O2 sensing solutions, their analytical features, and describe how they can be integrated in the current paradigm of biomedical research. Representative examples of the use of such systems in complex physiological studies with advanced tissue and disease models are given, in which they provide strict environmental control of dissolved and gaseous O2 (macroscopically and microscopically, by point measurements and high-resolution imaging in 2D and 3D), and important information about cellular function and changes in tissue metabolism under different conditions and treatments.