Toxicity testing plays an essential role in drug development. The primary objective of toxicology studies at preclinical stages is to evaluate the safety of drug candidates before human trials. This chapter provides a comprehensive review of the principles, methodologies, and regulatory requirements that ensure the safety of new therapeutic agents. Beginning with basic concepts, the chapter reviews definitions of dose-response relationship, route of exposure, metabolism and bioactivation variability, and risk assessment. Most commonly conducted animal studies to support drug development, including acute, subacute, chronic, repeat dose toxicity studies, are also discussed. Key topics including basic principles of development and reproductive toxicity and carcinogenicity risk assessment will be discussed. In the second half of the chapter, in vitro technologies such as various liver, heart, skin, and kidney models that can potentially support early hazard identification and derisking for drug candidates are discussed. Regulatory guidelines from major agencies like the FDA, EMA, and ICH are also discussed throughout the chapter, providing a framework for the design and interpretation of toxicology studies. Overall, the chapter illustrates strategies and comment challenges for mitigating potential toxicities, ultimately emphasizing the crucial contribution of toxicology to the safe and effective development of new drugs.