The detailed knowledge of mammalian cell metabolism and its adjustments to different cell properties and perturbations, such as disease and drug exposure, is of enormous value in the deeper understanding of pathological processes and drug mechanisms, as well as in the development of new and improved methods for diagnosis, follow-up of disease progression and treatment response. This review covers recent developments in the use of NMR-based metabonomics to characterize cellular metabolomes and interpret them in terms of metabolic changes taking place in a wide range of situations. The analytical methodology available is briefly presented and the applications developed so far are reviewed. These include differences in cell properties (e.g., drug resistance, cell cycle stage, specific growth conditions and genetic characteristics) and changes induced in response to different perturbations (e.g., disease, drug exposure and irradiation).