The supply of oxygen (O-2) to respiring tissue, cells, and mitochondria regulates metabolism, gene expression, and cell fate. Depending on the cell type and mitochondrial function, O-2 gradients between extra- and intracellular compartments may vary and play important physiological roles such as the regulation of activity of prolyl hydroxylases and adaptive responses to hypoxia. Here we present a new methodology for the analysis of localized O-2 gradients in cultures of adherent cells, using three phosphorescent Pt-porphyrin based probes with different localization. One new O-2 probe targeted to the cell membrane was developed and used together with existing MitoXpress and Nano2 probes to monitor mean pericellular (PC), extracellular (EC), and intracellular (IC) O-2 concentrations, respectively. Mouse fibroblasts and neuronal PC12 cells cultured in standard microplates were stained with probes and measured on a commercial time-resolved fluorescence reader in phosphorescence lifetime mode. Respiring cells exposed to various levels of atmospheric O-2 showed differences in oxygenation of their IC, PC, and EC compartments. Experiments with different cell numbers and modulation of respiration activity demonstrated that these gradients are dynamic and regulated by the O-2 diffusion and consumption rate. The new method facilitates the assessment of such gradients.