A new rapid, sensitive and convenient procedure is presented allowing determination of dUTPase activity. With [5-(3)H]dUTP used as the substrate, dUTPase, converts it to the corresponding monophosphate and is coupled with thymidylate synthase-catalyzed reaction, resulting in tritium release from [5-(3)H]dUMP. Following charcoal absorption of the labeled nuleotides, radioactivity of tritiated water is determined. The new assay was tested to show comparable results with a previously described assay, based on measuring dUTPase-catalyzed [5-(3)H]dUMP production.