This paper demonstrates that matrix-assisted laser desorption/ionization and time-of-flight mass spectrometry (MALDI-TOF MS) is very well suited for characterizing self-assembled monolayers (SAMs) of alkanethiolates on gold and, in particular, for analyzing chemical reactions on monolayers. Three examples establish the generality of MALDI-TOF for this purpose. The first example characterizes the Diels-Alder reaction of a cyclopentadiene to maleimide groups of a monolayer. The second example characterizes the immobilization of glutamine tert-butyl ester to a carboxylic acid terminated monolayer and subsequent removal of the tert-butyl group. The third example characterizes the exchange of alkanethiolates in a monolayer with alkanethiols in a contacting solution. The availability of commercial instruments for MALDI-TOF MS and the straightforward use of this technique for characterizing SAMs will make this an important analytical technique for programs that prepare and apply model substrates in chemistry and materials science.