The inventors have developed a rapid and sensitive fluorescence-based assay to quantify dNTPs. This assay relies on the principle that incorporation of a limiting dNTP is required for primer-extension and polymerase-mediated 5-3′ exonuclease hydrolysis of a quenched fluorophore-labeled probe resulting in fluorescence. The concentration of limiting dNTPs is directly proportional to the fluorescence generated. This assay has important applications in research that investigates the influence of pathological conditions or pharmacological agents on dNTP biosynthesis and regulation.