Background Expanding the cooperation and enlarging the participation of more diverse stakeholders within innovation ecosystems will increase their efficiency and capacity to contribute at local, regional, and national levels. Objective This paper presents the protocol for a systematic review that will identify “opening-up” strategies of innovation ecosystems for increasing the participation of more diverse innovation stakeholders, particularly from low-innovation countries, during the ecosystem formation period. Methods An algorithmic search in 4 databases (Web of Science, Cochrane Library, Scopus, and Social Science Research Network) will be applied based on the PerSPecTIF (perspective, setting, phenomenon of interest/problem, environment, optional comparison, time/timing, and findings) methodology, the Cochrane guidelines for qualitative evidence synthesis, and the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. Selection criteria for eligibility include peer-reviewed articles published after December 31, 1999, and containing original data. No restrictions will be placed on the article language and study region, design, or methodology. Methodological strengths and limitations will be assessed using the Critical Appraisal Skills Programme tool. The thematic synthesis method will be adopted, and the GRADE-CERQual tool will be used to assess confidence. Results A preliminary search in Web of Science revealed 2758 records. This work is part of the ANGIE project, which was funded by the European Union’s Horizon 2020 research and innovation program (grant 952152) in January 2021. We anticipate that the results of this systematic review will be published in spring 2022. Conclusions We anticipate that the outcomes of this systematic review will outline the best practices used by initiatives and networks, as well as their impacts on creating larger and more inclusive ecosystems. Trial Registration OSF Registries osf.io/hc62k 10.17605/OSF.IO/HC62K International Registered Report Identifier (IRRID) PRR1-10.2196/34071