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Background: The aim of this study was to develop and validate a liquid chromatography–tandem mass spectrometry (LC-MS/MS) assay for the measurement of uracil (U) and dihydrouracil (UH2) concentrations in dried saliva spots (DSSs), for the evaluation of dihydropyrimidine dehydrogenase (DPD) enzyme activity. Results: Nine 18-mm diameter DSS discs were extracted with acetate:isopropyl alcohol (85:15, vol/vol) and analyzed by LC-MS/MS. The assay was linear in the range of 10–1000 ng·mL−1, with accuracy between 89% and 112% and precision between 5.7% and 13%. The metabolic ratio [UH2]/[U] was stable in DSS for up to 9 days at 45°C. Concentrations of U and UH2, as well as the metabolic ratio, were highly concordant between matrices. Using a metabolic ratio classification cutoff of 1.16 for the identification of slow DPD metabolizers, 98.7% concordance was achieved between SS and saliva. Conclusions: DSS samples could be a useful alternative for DPD activity screening, particularly in locations with limited access to highly equipped laboratories.