The Humane World for Animals and IABS have been collaborating together since the 2019 IABS conference “Animal Testing for Vaccines Implementing Replacement, Reduction and Refinement: Challenges and Priorities” (Akkermans et al.) and under the AFSA Collaboration since 2020. They successfully organized online and face to face meetings (AFSA Collaborations Events) to promote dialogue and joint actions on animal testing replacement for both human and veterinary vaccines. This hybrid conference wishes to bring together again all the stakeholders in the field with the scope to concretely discuss about future projects and policy changes that need to be implemented to successfully phase out the reliance on animal testing within the vaccines testing control strategy (including product development, CMC and release), including the importance of vaccines’ supply chain sustainability and continuity and the benefits of this in supporting the role of vaccines in One Health (ensuring animal health and food chain security, addressing zoonosis and emergency disease situations as well as the critical role of vaccines in reducing AMR).