Mismatch repair corrects biosynthetic errors generated during DNA replication. Mismatch repair deficiency causes a mutator phenotype and directly underlies hereditary nonpolyposis colorectal cancer and some sporadic cancers. Because of remarkably high conservation of the mismatch repair machinery between the budding yeast (Saccharomyces cerevisiae) and humans, the study of mismatch repair in yeast has provided tremendous insights into the mechanisms of this repair pathway in humans. Here we describe a set of practical protocols for how to prepare the yeast and HeLa cell-free nuclear extracts and site-specific DNA mismatch substrates, and how to carry out the in vitro mismatch repair assay. We validated the yeast cell-free system by the mismatch repair deficient strain (Δmsh2) and the complementation assay with purified yeast MutSα.