Thermal desorption high-resolution mass spectrometry (TD-HRMS) is examined as a characterization tool for mixed self-assembled monolayers (SAMs) on gold. TD-HRMS provides a novel spectral view of the adsorption state of thiol and dithiol on gold, and it is confirmed in this study that thiolates, not thiols, are formed on the surface of gold by chemical bonding. Mass shifts in the TD-HRMS spectra indicate the number of Au-S bonds per thiol molecule and allow the accurate identification of the molecular formula of both single and mixed SAMs. The technique is also successfully used to observe the symmetric and asymmetric dimerization between constituent molecules of the SAM on gold. TD-HRMS is shown to provide information regarding the thermal desorption behavior of mixed SAMs, with clear distinctions between chemisorbed molecules and physisorbed molecules. TD-HRMS is demonstrated to be a useful means of estimating the surface molar ratio of mixed SAMs in that the molar ratio of mixed SAMs on gold is directly reflected in the obtained molecular ion intensity of constituent alkanethiols.