Human uracil DNA glycosylase (UNG2) is an enzyme whose primary function is to remove uracil bases from genomic DNA. UNG2 activity is critical when uracil bases are elevated in DNA during class switch recombination and somatic hypermutation, and additionally, UNG2 affects the efficacy of thymidylate synthase inhibitors that increase genomic uracil levels. Here, we summarize the enzymatic properties of UNG2 and its mitochondrial analog UNG1. To facilitate studies on the activity of these highly conserved proteins, we discuss three fluorescence-based enzyme assays that have informed much of our understanding on UNG2 function. The assays use synthetic DNA oligonucleotide substrates with uracil bases incorporated in the DNA, and the substrates can be single-stranded, double-stranded, or form other structures such as DNA hairpins or junctions. The fluorescence signal reporting uracil base excision by UNG2 is detected in different ways: (1) Excision of uracil from end-labeled oligonucleotides is measured by visualizing UNG2 reaction products with denaturing PAGE; (2) Uracil excision from dsDNA substrates is detected in solution by base pairing uracil with 2-aminopurine, whose intrinsic fluorescence is enhanced upon uracil excision; or (3) UNG2 excision of uracil from a hairpin molecular beacon substrate changes the structure of the substrate and turns on fluorescence by relieving a fluorescence quench. In addition to their utility in characterizing UNG2 properties, these assays are being adapted to discover inhibitors of the enzyme and to determine how protein–protein interactions affect UNG2 function.