Measuring tissue oxygenation in vivo is of interest in fundamental biological as well as medical applications. One minimally invasive approach to assess the oxygen partial pressure in tissue (pO(2)) is to measure the oxygen-dependent luminescence lifetime of molecular probes. The relation between tissue pO(2) and the probes' luminescence lifetime is governed by the Stern-Volmer equation. Unfortunately, virtually all oxygen-sensitive probes based on this principle induce some degree of phototoxicity. For that reason, we studied the oxygen sensitivity and phototoxicity of dichlorotris(1, 10-phenanthroline)-ruthenium(II) hydrate [Ru(Phen)] using a dedicated optical fiber-based, time-resolved spectrometer in the chicken embryo chorioallantoic membrane. We demonstrated that, after intravenous injection, Ru(Phen)'s luminescence lifetime presents an easily detectable pO(2) dependence at a low drug dose (1 mg/kg) and low fluence (120 mJ/cm(2) at 470 nm). The phototoxic threshold was found to be at 10 J/cm(2) with the same wavelength and drug dose, i.e., about two orders of magnitude larger than the fluence necessary to perform a pO(2) measurement. Finally, an illustrative application of this pO(2) measurement approach in a hypoxic tumor environment is presented. (C) 2014 Society of Photo-Optical Instrumentation Engineers (SPIE)