Global biodiversity decline is significantly driven by unsustainable value chains across multiple economic sectors. The EU Horizon project BIOTRAILS developed and tested a framework for the identification of pathways towards transformative change in biodiversity-relevant value chains. This report focuses on describing the methodologies applied and summarises the findings from the project. The framework combines the Leverage Points Framework with the AttitudesFacilitators-Infrastructure (AFI) Framework through a three-stage participatory methodology, integrating systematic literature reviews with stakeholder engagement. Applied to four diverse value chains – cocoa production in Peru, forest-based cultural products in Brazil, fisheries and aquaculture in Greece, and artisanal small-scale gold mining in Ghana – the methodology demonstrates that strategic intervention points can be systematically identified and contextualised when the framework is adapted to local environmental, social, economic, and institutional conditions. Despite differences in sectors, geographies, and sustainability challenges, the consistent methodological approach enabled the identification of both common patterns and context-specific leverage points across all case studies. This report provides practical guidance, tools, and lessons learned for practitioners, policymakers, and researchers seeking to apply the framework to other value chains, demonstrating that the methodology is effective when tailored to the specific contexts.