Altered metabolism is one of the defining features of cancer. Since the discovery of the Warburg effect in 1924, research into the metabolic aspects of cancer has been reinvigorated over the past decade. Metabolomics is an invaluable tool for gaining insights into numerous biochemical processes including those related to cancer metabolism and metabolic aspects of other diseases. The combination of untargeted and targeted metabolomics approaches has greatly facilitated the discovery of many cancer biomarkers with prognostic potential. Using mass spectrometry-based stable isotope-resolved metabolomics (SIRM) with isotopic labeling, a powerful tool used in pathway analysis, researchers have discovered novel cancer metabolic pathways and metabolic targets for therapeutic application. Metabolomics technologies provide invaluable metabolic insights reflecting cancer progression in coordination with genomics and proteomics aspects. The systematic study of metabolite levels in the metabolome and their dynamics within a biological organism has been, in recent years, applied across a wide range of fields. Metabolomics technologies have been applied to both early clinical trials and pre-clinical research in several essential aspects of human health. This chapter will give an overview of metabolomics technologies and their application in the discovery of novel pathways using isotopic labeled and non-labeled metabolomics.