This Guide aims to support the RPOs in fostering sustainable and inclusive Open Science ecosystem aligned with the European Commission’s broader objectives for research excellence, innovation, and collaboration. It is designed as a practical tool for decision-makers to establish a robust Open Science ecosystem within their RPOs, adapted to different institutional contexts. Its structured yet flexible approach allows RPOs to align their strategies with internal priorities and the broader Open Science landscape. The iterative, dynamic, and adaptable process recognizes diversity of institutional structures, and encourages RPOs to adapt the OS strategy to their organizational culture and goals while ensuring coherence with global Open Science trends, including UNESCO guidelines and EOSC developments and in alignment with the objectives of the ERA. Please note that this Guide on OS Strategy Development is connected to the Guide on OS Training.