The use of oxygen by cells is an essential aspect of cell metabolism and a reliable indicator of viable and functional cells. Viable and functional cells are essential for optimizing the therapeutic dose for cell therapy, tissue engineering, drug development, and many other biological processes and products. However, currently, there is no method to assess the cell metabolic activity nondestructively in 3D space and longitudinally as cells proliferate, metabolize, differentiate, or die. Here, we report partial pressure oxygen (pO₂) mapping of live cells as a reliable indicator of viable and metabolically active cells. For pO₂ imaging, we utilized trityl OX071-based pulse electron paramagnetic resonance oxygen imaging (EPROI), in combination with a 25 mT EPROI instrument, JIVA-25™, that provides 3D oxygen maps in tissues with high spatial and temporal resolution. To perform oxygen imaging in an environment-controlled apparatus using a standard biological lab consumable, that is, a multi-well plate, we developed a novel multi-well-plate incubator-resonator (MWIR) system that could accommodate 3 strips from a 96-well strip-well plate and image the middle 12 wells noninvasively and simultaneously. The MWIR system was able to keep a controlled environment (temperature at 37 °C, relative humidity between 70% - 100%, and a controlled gas-flow environment) during oxygen imaging and could keep cells alive for up to 24 h of measurement, providing a rare previously unseen longitudinal perspective of 3D cell metabolic activities. The robustness of MWIR was tested using an adherent cell line (HEK-293 cells), a nonadherent cell line (Jurkat cells), a cell-biomaterial construct (Jurkat cells seeded in a hydrogel), and a negative control (dead HEK-293 cells). Using MWIR, we demonstrate that EPROI is a versatile and robust method that can be utilized to observe the cell metabolic activity nondestructively, longitudinally, and in 3D. For the first time, we demonstrated that oxygen concentration in a multi-well plate seeded with live cells is inversely proportional to the cell seeding density, even if the cells are exposed to incubator-like gas conditions (95% air and 5% CO₂). Additionally, for the first time, we also demonstrate 3D, longitudinal oxygen imaging can be used to assess cells seeded in a hydrogel scaffold. These results demonstrate nondestructive, longitudinal 3D assessment of metabolic activities of cells using EPROI during 2D planar culture and during culture in a 3D scaffold system. The MWIR and EPROI approach may be useful for characterizing cell therapies, tissue engineered medical products and other advanced therapeutics.