AbstractThe present article reviews the mass spectrometric fragmentation processes and fragmentation energetics of leucine enkephalin, a commonly used peptide, which has been studied in detail and has often been used as a standard or reference compound to test novel instrumentation, new methodologies, or to tune instruments. The main purpose of the article is to facilitate its use as a reference material; therefore, all available mass spectrometry‐related information on leucine enkephalin has been critically reviewed and summarized. The fragmentation mechanism of leucine enkephalin is typical for a small peptide; but is understood far better than that of most other compounds. Because ion ratios in the MS/MS spectra indicate the degree of excitation, leucine enkephalin is often used as a thermometer molecule in electrospray or matrix‐assisted laser desorption ionization (ESI or MALDI). Other parameters described for leucine enkephalin include collisional cross‐section and energy transfer; proton affinity and gas‐phase basicity; radiative cooling rate; and vibrational frequencies. The lowest‐energy fragmentation channel of leucine enkephalin is the MH+ → b4 process. All available data for this process have been re‐evaluated. It was found that, although the published Ea values were significantly different, the corresponding Gibbs free energy change showed good agreement (1.32 ± 0.07 eV) in various studies. Temperature‐ and energy‐dependent rate constants were re‐evaluated with an Arrhenius plot. The plot showed good linear correlation among all data (R2 = 0.97), spanned over a 9 orders of magnitude range in the rate constants and yielded 1.14 eV activation energy and 1011.0 sec−1 pre‐exponential factor. Accuracy (including random and systematic errors, with a 95% confidence interval) is ±0.05 eV and 10±0.5 sec−1, respectively. The activation entropy at 470 K that corresponds to this reaction is −38.1 ± 9.6 J mol−1 K−1. We believe that these re‐evaluated values are by far the most accurate activation parameters available at present for a protonated peptide and can be considered as “consensus” values; results on other processes might be compared to this reference value. © 2010 Wiley Periodicals, Inc., Mass Spec Rev 30:298–320, 2011