Dihydropyrimidine dehydrogenase (DPD) is a key enzyme in the metabolic catabolism of chemotherapeutic agent 5-fluorouracil (5FU) and its derivatives, including capecitabine. Numerous genetic mutations have been identified in the DPD gene locus (DPYD), with a few key variants having functional consequences on enzymatic activity. Deficiencies in DPD activity have been shown to cause 5FU-treated cancer patients to experience severe drug-related toxicities, often requiring extensive medical intervention. We review the performance of assays that assess DPD and DPYD status, with an emphasis on the robustness for routine clinical applications. None of the current strategies are adequate to mandate routine DPD testing prior to starting a fluoropyrimidine-based therapy. However, further research and technological improvements will hopefully allow prospective identification of potentially toxic patients, in order to reduce the number of patients with severe, life-threatening side effects to 5FU treatment.