With the discovery that the hereditary cancer susceptibility disease Lynch syndrome (LS) is caused by deleterious germline mutations in the DNA mismatch repair (MMR) genes nearly 20 years ago, genetic testing can now be used to diagnose this disorder in patients. A definitive diagnosis of LS can direct how clinicians manage the disease as well as prevent future cancers for the patient and their families. A challenge emerges, however, when a germline missense variant is identified in a MMR gene in a suspected LS patient. The significance of a single amino acid change in these large repair proteins is not immediately obvious resulting in them being designated variants of uncertain significance (VUS). One important strategy for resolving this uncertainty is to determine whether the variant results in a non-functional protein. The ability to reconstitute the MMR reaction in vitro has provided an important experimental tool for studying the functional consequences of VUS. However, beyond this repair assay, a number of other experimental methods have been developed that allow us to test the effect of a VUS on discrete biochemical steps or other aspects of MMR function. Here, we describe some of these assays along with the challenges of using such assays to determine the functional consequences of MMR VUS which, in turn, can provide valuable insight into their clinical significance. With increased gene sequencing in patients, the number of identified VUS has expanded dramatically exacerbating this problem for clinicians. However, basic science research laboratories around the world continue to expand our knowledge of the overall MMR molecular mechanism providing new opportunities to understand the functional significance, and therefore pathogenic significance, of VUS.