Biallelic germline mutations in the base excision repair enzyme gene MUTYH lead to multiple colorectal adenomas and carcinomas referred to as MUTYH-associated polyposis. MUTYH removes adenine misincorporated opposite the DNA oxidation product, 8-oxoguanine (OG), thereby preventing accumulation of G:C to T:A transversion mutations. The most common cancer-associated MUTYH variant proteins when expressed in bacteria exhibit reduced OG:A mismatch affinity and adenine removal activity. However, direct evaluation of OG:A mismatch repair efficiency in mammalian cells has not been assessed due to the lack of an appropriate assay. To address this, we developed a novel fluorescence-based assay of OG:A repair and measured the repair capacity of MUTYH-associated polyposis variants expressed in Mutyh-/- mouse embryonic fibroblasts (MEFs). The repair of a single site-specific synthetic lesion in a green fluorescent protein reporter leads to green fluorescent protein expression with co-expression of a red fluorescent protein serving as the transfection control. Cell lines that stably express the MUTYH-associated polyposis variants G382D and Y165C have significantly lower OG:A repair versus wild-type MEFs and MEFs expressing human wild-type MUTYH. The MUTYH allele that encodes the Q324H variant is found at a frequency above 40% in samples from different ethnic groups and has long been considered phenotypically silent but has recently been associated with increased cancer risk in several clinical studies. In vitro analysis of Q324H MUTYH expressed in insect cells showed that it has reduced enzyme activity similar to that of the known cancer variant G382D. Moreover, we find that OG:A repair in MEFs expressing Q324H was significantly lower than wild-type controls, establishing that Q324H is functionally impaired and providing further evidence that this common variant may lead to increased cancer risk.