Metabolic profiling is commonly employed to investigate the global metabolic alterations of malignant cells or tissues. In the latter setting, neoplastic lesions are separated from adjacent, healthy tissues and their metabolites are quantified upon a chromatographic run coupled to mass spectrometry. Changes in the abundance of specific metabolites are then mapped on metabolic networks and the underlying metabolic circuitries are investigated as potential targets for the development of novel anticancer drugs. This approach, however, does not take into account the intrinsic heterogeneity of neoplastic lesions, which contain a large amount of non-transformed cells. To circumvent this issue, techniques have been developed that allow for the imaging of metabolites at the single-cell level. Here, we summarize established protocols that are suitable for imaging metabolites in animal cells (be them malignant or not) as well as in plant and prokaryotic cells. These methods are relevant for the study of the metabolic alterations that accompany oncogenesis and tumor progression. © 2014 Elsevier Inc. All rights reserved.