Considerable progress has been made in understanding the origins of genomic uracil and its role in genome stability and host defense; however, the main question concerning the basal level of uracil in DNA remains disputed. Results from assays designed to quantify genomic uracil vary by almost three orders of magnitude. To address the issues leading to this inconsistency, we explored possible shortcomings with existing methods and developed a sensitive LC/MS/MS-based method for the absolute quantification of genomic 2'-deoxyuridine (dUrd). To this end, DNA was enzymatically hydrolyzed to 2'-deoxyribonucleosides and dUrd was purified in a preparative HPLC step and analyzed by LC/MS/MS. The standard curve was linear over four orders of magnitude with a quantification limit of 5fmol dUrd. Control samples demonstrated high inter-experimental accuracy (94.3%) and precision (CV 9.7%). An alternative method that employed UNG2 to excise uracil from DNA for LC/MS/MS analysis gave similar results, but the intra-assay variability was significantly greater. We quantified genomic dUrd in Ung(+/+) and Ung(-/-) mouse embryonic fibroblasts and human lymphoblastoid cell lines carrying UNG mutations. DNA-dUrd is 5-fold higher in Ung(-/-) than in Ung(+/+) fibroblasts and 11-fold higher in UNG2 dysfunctional than in UNG2 functional lymphoblastoid cells. We report approximately 400-600 dUrd per human or murine genome in repair-proficient cells, which is lower than results using other methods and suggests that genomic uracil levels may have previously been overestimated.