Objective Photosensitization by singlet oxygen is a chemical reaction with cellular quenchers, mainly proteins. Therefore, optical detection of singlet oxygen in cells affects the investigated cells and potentially, falsifies the observed results of photosensitization. For this reason, the illumination intensity has to be kept low during a measurement to avoid killing and destruction of the investigated cells.Material & Methods The individual illumination dose for cells under investigation are reduced by distributing it among a large number of adherent growing cells. Adherent cell lines were investigated with regard to singlet oxygen production by means of simultaneous fiber-coupled irradiation at 635 nm and singlet oxygen phosphorescence detection using a specified photomultiplier tube in combination with the TCMPC counting system. Singlet oxygen kinetics could be measured in two different cell types incubated with different photosensitizers, by scanning a major part of the area of a confluent layer of cells in six well plates.Result In each case, singlet oxygen signals kinetics could be observed and analyzed. Incubation with increasing amounts of photosensitizer resulted in both, stronger signals and longer singlet oxygen decay times.Conclusion Focused excitation and observation in combination with scanning across the whole well results in successful determination of singlet oxygen kinetics from incubated adherent cells without detaching them from the well plate. Further development is required to reduce artifacts, likely originating from the plate bottom.