A new method for preparing phase-separated binary self-assembled monolayers (SAMs) of alkanethiols on gold is proposed. Binary SAMs of 3-mercaptopropionic acid (MPA) and hexadecanethiol (HDT) at various mixing ratios which exhibit phase separation in nanometer scale are used as starting substrates. Because the MPA domains in the binary SAMs have a less negative desorption potential, they can be selectively removed by controlling the potential of gold substrates in 0.5 M KOH. Other alkanethiols with various chain lengths and terminal groups can subsequently be adsorbed where MPA was originally adsorbed, resulting in phase-separated binary SAMs with different combinations of alkanethiols.