The in vitro DNA mismatch repair (MMR) assay is a very useful technique for studying the functions and the mechanisms of the MMR system in genome maintenance. This assay has been effectively used to evaluate MMR proficiency in various tumor cells and to identify the majority of the protein components required for MMR. The procedure for setting up and performing the MMR assay involves mismatch substrate preparation, cell extract preparation, and the repair assay. In this chapter, we describe the detailed methods for this functional in vitro assay.