The One Health Legislative Assessment Tool for Antimicrobial Resistance (AMR) (the Tool) has been developed by the legal teams of FAO, WHO and WOAH, with inputs from UNEP, through a process of extensive consultation with a range of legal experts across multiple disciplines. The Tool is currently being implemented in four countries prior to a wider rollout and implementation. The Tool assists countries in the identification of regulatory gaps relevant for AMR, and enables the prioritization of solutions through legal reform. It facilitates the examination of a range of legislative instruments and governance arrangements at local, region/province and national levels. Adopting a One Health approach that integrates and sustainably balances the health of people, animals and ecosystems, the Tool targets key regulatory elements in the following sectors (1) AMR governance (2); human health, (3) animal health and production, (4) food safety, (5) pesticide management, (6) plant health and (7) the environment. Under the Tool, cross-cutting themes and the need for multidisciplinary perspectives are emphasized to ensure multisectoral coordinated action against AMR. This Tool is currently being implemented in four countries prior to a wider rollout and implementation. While legislation is just one facet of the range of AMR responses recommended at international and national levels, legislation is often important or essential for many actions identified as a priority in National Actions Plans for AMR. The implementation of the Tool shows how legislation is used to introduce a One Health approach to manage AMR responses. A One Health approach to AMR requires a multidisciplinary response, among them multisectoral coordination mechanisms. In addition to bringing together a range of stakeholders as part of its development process, legislation formalizes and strengthens governance and accountability at local, regional and national levels within and across sectors and actors.