5-Fluorouracil (5-FU) is one of the most commonly used chemotherapeutics in the treatment of malignancies originating from breast, prostate, ovarian, skin and gastrointestinal tissues. Around 80% of administered dose of 5-FU is catabolized by dihydropirymidine dehydrogenase (DPD). Patients, in whom a deficiency or insufficient activity of this enzyme is observed, are at great risk of development of severe, even lethal, 5-FU toxicity. According to recent studies, so far over 30 mutations of DPYD gene, which are associated with DPD deficiency/insufficiency, have already been discovered. Currently, there are several analytical methods used for measurements of DPD activity. However, in this paper we report a novel, simple, economical and more accessible spectrophotometric method for measurements of DPD activity in the peripheral blood mononuclear cells (PBMCs) that was developed and validated on analysis of 200 generally healthy volunteers aged 22-63. We present two spectrophotometric protocols in this study, and as a reference method we used already described reverse phase high-performance liquid chromatography (RP HPLC) analysis. Basing on our findings, we conclude that spectrophotometric methods may be used as a screening protocol preceding 5-FU-based chemotherapy. Nevertheless, before introduction into clinical reality, our results should be confirmed in further larger studies.