During the 20th century, as drug products were being developed to treat both known and emerging human diseases and conditions, determining the safety of these new chemicals became of increasing importance and necessity. For a time, the safety of use in human populations was of question, let alone whether the drug product was truly effective. As such, US and international regulatory agencies have played a major role in establishing standardized testing to evaluate the safety and efficacy of drug products. Pharmacologic and toxicologic evaluation of a new drug in animals is an important part of the pharmaceutical development process prior to its first-time use in humans, as well as its potential chronic use in affected populations. Just as both science and technology have evolved over the past century and further, so have the guidelines that have been put forth to adequately and efficiently evaluate the toxicity of new drugs and their subsequent safety in humans. This review summarizes the historical highlights of the conduct of drug safety evaluations in animals, particularly with regard to chronic toxicity and carcinogenicity assessments, and how we have progressed to our current standards and protocols to ensure safe use of drug products in human populations.