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One Health addresses health challenges arising from the intertwined spheres of humans, animals and ecosystems. This handbook is the product of an interdisciplinary effort to provide science-based guidance for the evaluation of One Health and other integrated approaches to health. It guides the reader through a systems approach and framework to evaluate such approaches in a standardised way. It provides an overview of concepts and metrics from health and life sciences, social sciences, economics, and ecology that are relevant for the evaluation of the processes involved, as well as the characterisation of expected and unexpected outcomes of One Health initiatives. Finally, the handbook provides guidance and practical protocols to help plan and implement evaluations in order to generate new insights and provide meaningful information about the value of One Health. The handbook is intended for practitioners, researchers, evaluators as well as funders of integrated approaches to health and beyond.