Numerous studies have shown that select DNA repair enzyme activities impact response and/or toxicity of genotoxins, suggesting a requirement for enzyme functional analyses to bolster precision medicine or prevention. To address this need, we developed a DNA Repair Molecular Beacon (DRMB) platform that rapidly measures DNA repair enzyme activity in real-time. The DRMB assay is applicable for discovery of DNA repair enzyme inhibitors, for the quantification of enzyme rates and is sufficiently sensitive to differentiate cellular enzymatic activity that stems from variation in expression or effects of amino acid substitutions. We show activity measures of several different base excision repair (BER) enzymes, including proteins with tumor-identified point mutations, revealing lesion-, lesion-context- and cell-type-specific repair dependence; suggesting application for DNA repair capacity analysis of tumors. DRMB measurements using lysates from isogenic control and APE1-deficient human cells suggests the major mechanism of base lesion removal by most DNA glycosylases may be mono-functional base hydrolysis. In addition, development of a microbead-conjugated DRMB assay amenable to flow cytometric analysis further advances its application. Our studies establish an analytical platform capable of evaluating the enzyme activity of select DNA repair proteins in an effort to design and guide inhibitor development and precision cancer therapy options.