Four approaches have been explored for the preparation of maleimido-functionalized self-assembled monolayers (SAMs) on silicon. SAMs prepared by self-assembly of maleimido-functionalized alkyltrichlorosilanes (11-maleimido-undecyl-trichlorosilane) on oxide-covered silicon yield higher signals from maleimido functionalities in ATR-IR (attenuated total reflection IR) spectroscopy and XPS (X-ray photoelectron spectroscopy) than the other three methods. The surface composition of maleimido groups was tailored further by the formation of mixed monolayers with nonfunctionalized alkyltrichlorosilanes (decyltrichlorosilane). The order of the alkyl chains within the monolayers only slightly depends on the composition of the mixed monolayers. We utilized the maleimido-terminated SAMs to bind various nucleophilic compounds, alkylamines, alkylthiols, and thiol-tagged DNA oligonucleotides by means of conjugate addition.