Over the past century, most pandemics have been triggered by pathogens spilling over from animals to humans (zoonotic spillover). Global efforts to mitigate the risk of another pandemic predominantly focus on responding to pathogens after they are already spreading among humans. However, the COVID-19 pandemic highlighted the limitations of reactive measures; even stringent control measures cannot contain a rapidly spreading pathogen. Interventions that prevent pathogens from spilling over from wildlife into humans in the first place (primary prevention) benefit everyone globally and may have co-benefits for climate change and biodiversity conservation. A major barrier to primary prevention is a lack of clear, evidence-based best practices. To address this gap, we propose a collaboration with the Lancet-PPATS Commission on the Prevention of Viral Spillover, to develop the One Health framework for spillover investigation and primary pandemic prevention. We will facilitate the collection of high-quality evidence worldwide on the root causes of pathogen spillover by creating transdisciplinary, One Health-focused guidance for spillover investigations; develop innovative approaches for standardized, case-control site selection for spillover investigations, with an emphasis on ecological drivers; pursue an integrated communication strategy to disseminate findings and recommendations to public health practitioners, national governments, and international treaty organizations. We will provide input for the World Bank Pandemic Fund (WBPF) grants program, which aims to allocate over $3 billion to enhance pandemic prevention and preparedness in low- and middle-income countries. By enabling integration of investigations of ecological drivers of spillover into other routine clinical and epidemiologic investigation approaches, we seek to direct funds towards primary prevention and facilitate ecological solutions to spillover. Through this collaboration, we anticipate immediate global impact, with the potential for our approach to become a central strategy for generating the evidence that can underpin interventions for reducing pandemic risks, while simultaneously advancing nature conservation and climate mitigation.