An impressive percentage of biomedical knowledge and advances were achieved through animal research and cell culture investigations. For drug testing and disease researches, both animal models and preclinical trials with cell cultures are extremely important, but they present some limitations, such as ethical concern and lack of representatively of human tissues and organs. Most cells are currently cultured using two-dimensional (2D) methods, but new and improved methods that implement three-dimensional (3D) cell culture techniques suggest convincing evidence that much more advanced experiments can be performed with more complex information. The environment and cell types in 3D culture can be manipulated to mimic tissue in vivo and provide more accurate data on cell-to-cell interactions; the cultivation techniques are based on a scaffold, which can be based on hydrogel or polymeric material, in addition there are techniques without using scaffold, such as suspended microplates, magnetic levitation and microplates for spheroids with ultra-low fixation coating. Even though 3D culturing is clearly incapable of replacing other current research types, they might continue to replace some unnecessary animal experimentation, as well as improve monolayer cultures. It is not even recommended or expected that 3D models substitute all other research types, but in regard to animal testing, they come in hand for the 3 Rs: Reduction, Refinement, Replacement. In this aspect, 3D culture emerges as valuable alternatives to the investigation of functional, biochemical and molecular aspects of human pathologies.