High-resolution electron energy loss spectroscopy (HREELS) as a probe of self-assembled monolayers (SAMs) obtained from thiols is here reviewed. It is shown how this surface selective spectroscopy can deliver information about these molecular hybrid systems. The layer ordering and organization into domains is probed through the analysis of the broadening of the elastic peak. Interfacial bonding between the thiol group and the substrate as well as the orientation of different parts of the grafted molecule is studied through the analysis of the vibrations induced by incident electrons interacting by dipolar mechanisms. The nature of outermost molecular groups exposed to the vacuum is inferred through the interactions by electron impact. HREELS spectra can provide additional information on interband transitions, excitation thresholds, the values of the gap and, besides, about intermolecular interactions. The interest of HREELS for studying strands of DNA ordered in SAMs, as models of DNA, is evinced.