One Health is an interdisciplinary approach that advocates for programs and policies that integrate governance, conservation, agriculture, disease ecology, and global health to achieve desired health outcomes. However, rigorous research around integrated One Health programming is limited and/or in very early stages, especially concerning counterfactual-based studies focused on the effectiveness of integrated conservation and health programming, including those focused on the intersection of zoonosis spillover risk in the context of land-use change. We argue that filling these knowledge gaps requires an implementation science approach. This requires evaluation through a counterfactual lens, but also requires a new approach to donor funded program design and the entire project cycle. We present benefits, challenges, and lessons learned from three case studies of efforts at applying an implementation science approach to integrated conservation and health programming in Madagascar, Zambia, and Ghana and Côte d’Ivoire. We demonstrate the value of integrating an implementation science approach at program inception, and the importance of building the evidence base on the effectiveness of integrated conservation and health programming. We demonstrate that despite significant challenges, it is possible to pursue an implementation science approach for cross-sectoral conservation and health programs, including studies on zoonosis spillover risk in the context of efforts to improve environmental outcomes.