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A future bio-based economy envisions the transformation of the petrochemical industry into using biomass such as wood (waste) as a major resource. The early-stage evaluation of a biorefinery project requires the optimization of the lay-out of the supply chain considering the spatio-temporal variability of the availability of feedstock and the techno-economical characteristics of the biorefinery process. Therefore, the presented methodology was developed combining three models: (1) a forest management and planning tool providing a detailed prediction on the wood resource availability as well as the harvested feedstock quantity and cost with respect to location and time, (2) a techno-economic assessment model of the biorefinery process (e.g., species-specific conditions, capacity, CAPEX, OPEX), and (3) a strategic supply chain optimization model combining the insights of (1) and (2) into a spatio-temporal explicit supply chain analysis. The developed methodology has been evaluated through a case-study on the emerging reductive catalytic fractionation (RCF) biorefining in the Flanders region (EU) and shows that the most economically interesting configuration is one large biorefinery with a yearly wood chip intake of 150 kton. The biorefinery location reflects the available feedstock distribution in Flanders and is suggested to be situated best in the most forested region. The proposed methodology proved to be dynamic and robust: (1) input data and technical calculations can easily be adapted or updated; (2) the methodology can be applied to a broad range of applications beyond the scope of the biorefinery, to different feedstock choices; (3) the impact of the biorefinery location on e.g. energy balance, CO 2 emissions, and financial balance can be assessed. [Display omitted] • Integration of resource availability, techno-economic analysis, supply chain design. • Supports designing new bio-based value chains at low Technology Readiness Level. • Case study on emerging reductive catalytic fractionation in Flanders. • Region features and time variations impact process feasibility and chain layout. [ABSTRACT FROM AUTHOR]