In 2021, the American Association of Veterinary Medical College’s (AAVMC) Board of Directors established a Task Force to develop Guidelines on the Use of Animals in Veterinary Education. The Task Force consisted of representatives from nine AAVMC member schools from four countries across three continents. The Guidelines, published online in October 2022, offered recommendations for schools on how to improve their animal use policies, use of animal alternatives, and transparency.While the Guidelines provided overarching principles and broad recommendations, the next step was to write a handbook to accompany the Guidelines, elaborating on how institutions could implement the Guideline’s recommendations, enabling them to support and promote humane and ethical animal use, guided by the 4 Rs: replacement, reduction, refinement, and respect. The editors worked with internationally recognized co-authors to create the Handbook. The Handbook’s 11 chapters align with the recommendations made in the Guidelines. Authors describe and share best practices for acquisition and use of cadavers; use and management of live animals including small, large, and exotic and zoological species; and highlight where alternatives can be deployed in meeting educational outcomes.The Guidelines and the AAVMC Handbook on the Use of Animals in Veterinary Education allow educators worldwide to review and, where appropriate, adjust their veterinary programs’ approaches and policies. The Guidelines and Handbook should be interpreted as guiding principles and are not meant to be prescriptive. Individual institutions are encouraged to consider their own unique institutional, national and regional circumstances.