A simple, minimally invasive methodology for the analysis of intracellular oxygen in populations of live mammalian cells is described. Loading of the cells with the phosphorescent O2-sensing probe, MitoXpress, is achieved by passive liposomal transfer or facilitated endocytosis, followed by monitoring in standard microwell plates on a time-resolved fluorescent reader. Phosphorescence lifetime measurements provide accurate, real-time, quantitative assessment of average oxygen levels in resting cells and their alterations in response to stimulation. Analytical performance of the method is examined, optimized, and then demonstrated with different suspension and adherent cell lines including Jurkat, PC12, A549, HeLa, SH-SY5Y, and C2C12, by monitoring responses to mitochondrial uncouplers, inhibitors, plasma membrane depolarization, and Ca2+ effectors. The assay provides relevant, information-rich data on cellular function and metabolism. It allows monitoring of small, rapid, and transient changes in cell respiration and screening of new chemical entities.