Purpose <p>In 20–30% of the patients, fluoropyrimidines (5-FU) based chemotherapy leads to severe toxicity, which is associated with dihydropyridine dehydrogenase (DPD) deficiency. Therefore, <i>DPYD</i> genotyping became standard practice before treatment with fluoropyrimidines. Nevertheless, only 17% of the patients with severe toxicity have a <i>DPYD</i> variant. Therefore, an urgent need persists to investigate other strategies contributing to prediction and prevention of toxicity. Endogenous DPD substrates are considered as potential biomarkers to predict toxicity, yet contradictional data exist on demonstrating uracil as a reliable biomarker. Thymine as biomarker for toxicity has been investigated less. The aim of this study was to determine the association between the concentrations of uracil, thymine dihydrouracil (DHU) and dihydrothymine (DHT), with the systemic drug exposure of 5-FU and DPD enzyme activity in patients treated with 5-FU.</p> Methods <p>We included 36 patients with gastrointestinal malignancy who received 5-FU infusion. <i>DPYD</i> genotyping was conducted before start of treatment. Blood samples for determining 5-FU, uracil and thymine concentrations during infusion and DPD enzyme activity were taken.</p> Results <p>We found a significant correlation between the 5-FU systematic exposure and baseline thymine concentrations (<i>R</i>² = 0.1468; <i>p</i> = 0.0402). DPD enzyme activity was significantly correlated with baseline thymine concentrations but no correlation was found between DPD enzyme activity and 5-FU systemic drug exposure.</p> Conclusion <p>5-FU dose individualization based on thymine concentrations could be a promising addition to <i>DPYD</i> genotyping to predict 5-FU-induced toxicity. Larger prospective trials are needed to examine thymine as predictor for toxicity in daily practice.</p> Trial registration <p>Trial NL7539 at ‘Overview of Medical Research in the Netherlands’ (ID NL-OMON21471). Date of registration 19-02-2019.</p>