Metabolomics can play a particularly important role in elucidating novel anabolic and catabolic pathways in bacteria and fungi, and in understanding the dynamics of metabolism. In these approaches, an isotopically labelled substrate, with an artificially high abundance of isotopic label, is fed to the microorganism under study. The products become isotopically labelled, and can be measured using a combination of mass spectrometry and nuclear magnetic resonance spectroscopy. This mass isotopomer analysis is referred to as time and relative differences in systems (TARDIS)-based analysis, as it measures and quantifies the temporal sequential emergence of these labelled products. In this review, we cover this topic from an experimental point of view in relation to the study of metabolism, and summarise how the application of radioactive and stable isotopes is being used in pathway elucidation and metabolic flux determination (fluxomics).