<p>It is well established that malignant cells alter their metabolism to support proliferation, but the nutrients required to meet the anabolic demands of different cancers located at various anatomical sites throughout the body remain largely unknown. Moreover, the extent to which nutrients are supplied by neighbouring stromal cells or distant tissues, possibly due to metabolic reprogramming, is poorly understood. Metabolomics provides a unique biochemical approach to address these gaps in our knowledge, but cancer studies require careful consideration because it is challenging to identify appropriately matched control samples for comparison. Here, we detail a collection of metabolomics workflows designed to interrogate cancer across three discrete scales. First, we describe experiments to define the nutrient demands of cancer cells themselves. Second, we focus on identifying metabolic relationships between neighbouring cells in the tumour microenvironment. Finally, we highlight strategies to explore the metabolic crosstalk between cancer cells and distant tissues in the tumour macroenvironment. The approaches outlined span cells in culture, animal models and human specimens from patients with cancer. Special emphasis is dedicated to the application of emerging technologies and computational pipelines in the field of mass spectrometry that enable global profiling of metabolites and lipids.</p>