3D cell cultures can be used as alternatives to in vivo studies, aiming to more effectively adhere to the 3R (reduction, refinement, replacement) principle, which is based on reducing the number of animals used for research, improving experimental procedures and searching for alternative methods. The development and application of 3D cell cultures is not only a more ethical but also a more cost-effective alternative to in vivo animal models. Three-dimensional cell cultures can be used to form spheroids and organoids, which resemble functional organs and physiologically better reflect tissue architecture, cellular interactions, and the composition of extracellular matrix. The spheroid biological system has a well-defined structure, with an outer layer composed of proliferating cells and an inner core composed of hypoxic and necrotic cells. This heterogeneous cell system can be used for genotoxicity studies of various factors, modelling their penetration and distribution in various spheroid areas. The aim of these studies would be to select 3D cell systems that are most suitable for various genotoxicity tests and use them for genotoxicity assessment.