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Metabolomic data sets provide a direct read-out of cellular phenotypes andare increasingly generated to study biological questions. Our previous workrevealed the potential of analyzing extracellular metabolomic data in thecontext of the metabolic model using constraint-based modeling. Through thiswork, which consists of a protocol, a toolbox, and tutorials of two use cases,we make our methods available to the broader scientific community. The protocoldescribes, in a step-wise manner, the workflow of data integration andcomputational analysis. The MetaboTools comprise the Matlab code required tocomplete the workflow described in the protocol. Tutorials explain thecomputational steps for integration of two different data sets and demonstratea comprehensive set of methods for the computational analysis of metabolicmodels and stratification thereof into different phenotypes. The presentedworkflow supports integrative analysis of multiple omics data sets.Importantly, all analysis tools can be applied to metabolic models withoutperforming the entire workflow. Taken together, this protocol constitutes acomprehensive guide to the intra-model analysis of extracellular metabolomicdata and a resource offering a broad set of computational analysis tools for awide biomedical and non-biomedical research community.

MetaboTools: A comprehensive toolbox for analysis of genome-scale metabolic models