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Summary: Here, we present a protocol for analyzing the global metabolic landscape in breast tumors for the purpose of metabolism-based patient stratification. We describe steps for analyzing 1,454 metabolic genes representing 90 metabolic pathways and subjecting them to an algorithm that calculates the deregulation score of 90 pathways in each tumor sample, thus converting gene-level information into pathway-level information. We then detail procedures for performing clustering analysis to identify metabolic subtypes and using machine learning to develop a signature representing each subtype.For complete details on the use and execution of this protocol, please refer to Iqbal et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.