Aim: Fluoropyrimidines are commonly used anti-cancer drugs, but lead to severe toxicity in 10–30% of patients. Prospective DPYD screening identifies patients at risk for toxicity and leads to a safer treatment with fluoropyrimidines. This study evaluated the routinely application of prospective DPYD screening at the Leiden University Medical Center. Methods: Prospective DPYD screening as part of routine patient care was evaluated by retrospectively screening databases and patient files to determine genotype, treatment, dose recommendations and dose adjustments. Results: 86.9% of all patients with a first fluoropyrimidine prescription were screened. Fourteen out of 275 patients (5.1%) carried a DPYD variant and received a 25–50% dose reduction recommendation. None of the patients with a DPYD variant treated with a reduced dose developed toxicities. Conclusion: Prospective DPYD screening can be implemented successfully in a real world clinical setting, is well accepted by physicians and results in low toxicity.