Toxicity testing with animals is expensive, ethically controversial, and not always predictive of the human response. Cell-based assays are regarded as an alternative. However, conventional two-dimensional cell cultures do not reproduce the tissue architecture in vivo, and do not forecast organ-specific toxicity. On the other hand, three-dimensional cultures emulate the biochemistry and mechanics of the microenvironment in tissues more closely. Therefore, they address the limitations of both animals and two-dimensional cultures, and provide more accurate data on the effects of short- and long-term exposure to toxicants. We provide an up-to-date overview on the use of three-dimensional cell cultures in toxicology. We anticipate that three-dimensional cultures will become invaluable to accomplish the 3R agenda (refinement, reduction, and replacement) for animal-based toxicity testing and will play a major role for the Registration, Evaluation and Authorisation of Chemicals in the European Union (REACH legislation).