Chemical characterization of complex mixtures of large alkanes is critically important for many fields, including petroleomics and the development of renewable transportation fuels. Tandem mass spectrometry is the only analytical method that can be used to characterize such mixtures at the molecular level. Many ionization methods used in mass spectrometry involve proton transfer to the analyte. Unfortunately, very few proton affinity (PA) values are available for alkanes. Indeed, previous research has shown that most protonated alkanes (MH+) are not stable but fragment spontaneously via the elimination of a hydrogen molecule to form [M - H]+ ions. Here, the PAs of several n-alkanes and alkylcyclohexanes containing 5-8 carbon atoms, n-pentane, n-hexane, n-heptane, n-octane, cyclohexane, methylcyclohexane, and ethylcyclohexane, were determined via bracketing experiments by using a linear quadrupole ion trap mass spectrometer. Monitoring the formation of the [M - H]+ ions in reactions between the alkanes and protonated reference bases with known PAs revealed that the PAs of all the alkanes fell into the range 721 ± 20 kJ mol-1. In order to obtain a more accurate estimate of the relative PAs of different alkanes, two alkanes were introduced simultaneously into the ion trap and allowed to react with the same protonated reference base. Based on these experiments, the longer the alkyl chain in an n-alkane or alkylcyclohexane the greater the PA. Further, when considering alkanes with the same number of carbon atoms, the PAs of those with a cyclohexane ring were found to be greater than those with no such ring.