Current methods for measuring deoxyribonucleoside triphosphates (dNTPs), the building blocks of DNA, employ reagent, labor and time-intensive assays. We recently developed a rapid and sensitive 96-well fluorescence-based assay to quantify cellular dNTPs utilizing a standard real-time PCR thermocycler. Despite greatly improving the procedure and cost-effectiveness of dNTP measurement, this novel approach lacked the sensitivity to measure dNTPs in some difficult cell-based applications. We subsequently explored some preliminary modifications to the assay design and noted outstanding improvements to numerous aspects of assay performance including sensitivity. The goal of this proposal is to continue the development of this assay with the goal of establishing it as the gold standard for dNTP analysis worldwide.