A multidisciplinary and multisectoral collaboration, through a One Health approach is required to effectively prepare for, detect, assess, and respond to emerging and endemic zoonotic diseases. However, external and internal health system evaluations continue to identify major gaps in capacity to implement multisectoral and multidisciplinary collaboration within and between many countries, and countries are asking for support from the Tripartite to fill these gaps. This guide is the response to those requests.