In an NMR sample, precise measurement of the absolute absorption frequencies of nuclei is difficult. A standard internal reference compound is added, and the frequency difference between the reference signal and sample signals is measured.

The internal reference compound generally used in NMR spectroscopy is tetramethylsilane (TMS). TMS is preferred because it is chemically inert, soluble in NMR solvents, and easily removable. Also, the highly shielded methyl protons in TMS yield an intense signal at a lower frequency than most other organic molecules. Because of these advantages, TMS is used as a primary reference in proton, carbon, and silicon NMR spectroscopy. If a suitably inert reference compound is not available, the reference is kept in a capillary tube within the NMR tube and called an external reference.

In addition, deuterated NMR solvents such as CDCl₃, D₂O, and (CD₃)₂SO contain residual protons whose signal can be used as a secondary reference. Furthermore, the signal from the deuterium itself can be used to monitor the instrument's magnetic field by a technique called locking. During locking, the deuterium signal is constantly compared to a reference frequency and adjusted if there is any variation.