Metabolic profiling using mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) is integral to the rapidly expanding field of metabolomics, which is making progress in toxicology, plant science and various diseases, including cancer. In the area of oncology and metabolic phenotyping, researchers have probed the known changes in malignant cellular pathways using new experimental techniques to gain more insights, and others are exploiting these same cellular pathways for therapeutic drug targets and for novel cancer biomarkers, with the ultimate goal of translation to the clinic. Here, we discuss the challenges and opportunities in metabolic phenotyping for discovering novel cancer biomarkers, and we assess the clinical applicability of MS and NMR.