Abstract The term “nutraceuticals” has been used to describe products derived from natural sources that provide additional health benefits beyond the basic nutritional value in foods. These include dietary supplements, functional foods, medicinal foods, and “farmaceuticals.” It is believed that such products promote general well-being, control symptoms, and possibly prevent disease processes. The safety assessment of nutraceuticals is an essential part in their development for commercialization. To this end, several methods, both in vitro and in vivo, have been described in the literature. In this chapter, an attempt has been made to briefly describe in vitro models for safety and toxicity evaluation of nutraceuticals, potentially, as an alternative to conducting animal studies. These include evaluation of cytotoxicity, genotoxicity, major organ systems toxicity such as hepatotoxicity, gastrointestinal toxicity, cardiovascular toxicity, nephrotoxicity, and toxicity as determined using more recent “omics” and “in silico” approaches. Further, emerging technologies such as “human, organ, and/or lab in a chip” are also discussed. It is believed that, going forward, these in vitro alternatives will play a significant role in minimizing animal use and contribute toward the “3Rs” (reduce, replace, and refine) concept. However, it should be cautioned that such methodologies, as we know them thus far, still need to be validated sufficiently so they can play a major role as an alternate to in vivo testing. Currently, they contribute significantly toward understanding the mechanism of action of xenobiotics, including nutraceuticals. Additionally, they serve as initial screens in understanding the effects of test agents on complex biological systems while assisting in the evaluation of efficacy, safety, and toxicity during the development of products, including nutraceuticals.