During apoptosis several mitochondrial proteins are released. Some of them participate in caspase-independent nuclear DNA degradation, especially apoptosis-inducing factor (AIF) and endonuclease G (endoG). We studied the structure, cellular localization, and interactions of several proteins in silico and in vitro using fluorescent microscopy. Bioinformatic predictions were conducted to analyze the presence of interaction sites in the studied proteins. We conducted molecular modeling of proteins with unknown 3D structure: endonuclease G, CPS-6, WAH-1, and heat shock protein 70-1. These models were then refined by MolProbity server and employed together with experimentally known 3D structures of other proteins like AIF and cyclophilin A in molecular docking simulations of interactions. Fluorescence resonance energy transfer (FRET) technique and consequent image analysis was used to evaluate the interactions of fluorescently labeled proteins in cells. Our results represent new information about the structure, cellular localization, and interactions of several proteins involved in caspase-independent apoptotic death. This work was supported by The Ministry of Education, Youth and Sports of the Czech Republic (projects number MSM0021622419, 2B06052, and LC535).