Non-invasive optical techniques on the basis of quenching of phosphorescence by molecular oxygen represent a powerful tool to perform mapping of oxygen content in biological systems. The development of phosphorescent oxygensensitive probes suitable for biological applications, especially in vivo, remains a challenging task. In this paper we applied for biological investigations one novel and one previously published Ir(III)-based complexes. This complexes demonstrate low cytotoxicity at the concentrations ≤70 μM and good uptake by cultured cancer cells. Both compounds were primarily located within the cytoplasm. Using TCSPC-based phosphorescence lifetime macro-imaging in vivo, we found that IR-2 complex shows detectable phosphorescence in tumor tissue in mice upon local injection. These results illustrate the high potential of organometallic complexes under study for mapping of oxygen level in cells and tissues.