Thymidylate syntheses are essential for DNA biosynthesis and catalyzes the reductive methylation of dUMP (2’-deoxyuridine- 5’momphospate) to form dTMP (2’ deoxythymidine-5’-momphosphate). Humans rely on the thyA encoded classical Thymidylate syntheses whereas many human pathogens produce a novel type of Flavin-dependent Thymidylate synthases (FDTSs), which are encoded by the thyX gene. The molecular mechanism of catalysis differs between classical TS and FDTS, therefore making thyX coded proteins a suitable antimicrobial target. Although several studies have investigated FDTS catalysis, the kinetic mechanism, such as the order of substrate binding and release, is still currently under investigation. One of the current problems of constructing a complete kinetic model for a multiple substrate system is the amount of reaction conditions and time required for analyzing each reaction. The currently accepted 14C-dUMP assay is time-consuming and requires long analysis by High-Pressure Liquid Chromatography (HPLC). In our research study a tritium (3H) release assay is being developed to analyze multiple reaction conditions simultaneously, providing an efficient way to construct a multi-substrate kinetic model. Here we compare product formation indicated by the release of tritium from 5-3H-dUMP into the water to the accepted 14C-dUMP assay to validate this assay for following FDTS activity. Conditions were also optimized for treatment of samples in order to separate unreacted 5-3H-dUMP from titrated water without the use of HPLC, greatly shortening analysis time. This tritium release assay should allow the collection of numerous reaction rates required to construct a more complete kinetic model for FDTS enzymes. Kinetic studies will be important in resolving the binding and release order of substrates and products, and reveille the identity of enzymatic complexes, which in turn will aid in rational drug development.