Breast cancer is one of the most dangerous forms of cancer worldwide. Continuous research is therefore essential in order to find realistic treatment options. 3D tumor spheroids have been proven to imitate the structural and physiological TME of in vivo tumors relatively more accurate than the more widely used 2D cell cultures. During this study, the aim was to find the optimal conditions for single spheroid formation, employing breast cancer cells and utilizing GrowDex. Different concentrations and cell densities were used, as well as differing methods (GrowDex addition prior or (days) after cell cluster formation). The liquid overlay technique was used to grow the spheroids, which were observed for a maximum of 14-16 days. Finally, an ATP viability assay was performed to determine whether the spheroids contained a proliferation zone, which corresponds to active cells. Single spheroids were obtained for all cell densities. The relatively high GrowDex concentrations responded well to a cell density of 2000 cells, while the relatively low GrowDex concentrations responded well to cell densities of 5000 and 10000 cells. Through the ATP viability assay the samples illustrated the presence of a proliferation zone, and thus active cells.