We studied the cellular localization of the apoptotic proteins endonuclease G, AIF, and AMID in living cells using both single-cell colocalization image analysis and nuclear translocation image analysis. Image data were acquired using automated fluorescent microscope Zeiss Axiovert 200M with spinning disc confocal unit Yokogawa CSU-10. We confirmed the mitochondrial localization of endonuclease G and AIF by single-cell colocalization image analysis. We found the AMID protein to be cytoplasmic, most probably incorporated into the cytoplasmic side of the membranes of various organelles. The highest concentration of AMID was observed associated with the Golgi. Colocalization of AMID with lysosomes was also indirectly confirmed by analysis of AMID-rich vesicle velocity using manual tracking analysis. A novel analysis of time-lapse fluorescence image data during staurosporine-induced apoptosis revealed nuclear translocation only for endonuclease G and AIF. This work was supported by The Ministry of Education, Youth and Sports of the Czech Republic (projects number MSM0021622419 and LC535).