Carbon‐13 NMR spectroscopy in combination with ¹³C‐labeled substrate infusion is a powerful technique for measuring a large number of metabolic fluxes noninvasively in vivo. It has been used to quantify glycogen synthesis rates, establish quantitative relationships between energy metabolism and neurotransmission, and evaluate the importance of different substrates. Measurements can, in principle, be performed through direct ¹³C NMR detection or via indirect ¹H‐[¹³C] NMR detection of the protons attached to ¹³C nuclei. The choice of detection scheme and pulse sequence depends on the magnetic field strength, whereas substrate selection depends on metabolic pathways. ¹³C NMR spectroscopy remains a challenging technique that requires several nonstandard hardware modifications, infusion of ¹³C‐labeled substrates, and sophisticated processing and metabolic modeling. In this study, the various aspects of direct ¹³C and indirect ¹H‐[¹³C] NMR are reviewed with the aim of providing a practical guide. Copyright © 2011 John Wiley & Sons, Ltd.