Fluoropyrimidine drugs such as 5-fluorouracil (5-FU) and capecitabine are a mainstay in the treatment of numerous solid tumors, including colorectal cancers, alone or as part of combination therapies. Cytotoxic drugs such as 5-FU and oral capecitabine display narrow therapeutic indexes combined with high interpatient pharmacokinetic variability. As a result, severe toxicities often limit or delay the administration of successive, optimal chemotherapeutic courses, leading to unfavorable clinical outcome in patients with cancer. Catabolism and deactivation of fluoropyrimidine drugs depend on a single and exclusive enzymatic step driven by dihydropyrimidine dehydrogenase (DPD). Dihydropyrimidine dehydrogenase is prone to marked circadian rhythms, drug-drug interactions, and genetic polymorphisms; influence of its erratic activity on 5-FU pharmacokinetics and toxicity profile has been extensively investigated, and it is now well known that DPD deficiency leads to severe toxicities with 5-FU or possibly capecitabine exposure. With the ever-increasing number of patients with cancer likely to be treated with fluoropyrimidines, predicting and preventing the occurrence of such toxicities is now a major issue in clinical oncology. Early determination of DPD status in patients with cancer would allow identification of those at risk and help in subsequent dose adjustment or selection of other treatment modalities. Numerous methods, either genotypic or phenotypic, have been proposed to achieve this goal. This review covers a wide range of techniques available to establish DPD status in patients with cancer.