Background/Aim: Solar ultraviolet radiation (UVR) is a carcinogen and irradiation of the skin results in DNA damage. Cyclobutane pyrimidine dimers (CPDs), including thymidine dimers, are among the most frequent forms of DNA damage. When CPDs are formed, the nucleotide excision repair system is activated and CPDs are excreted in the urine. Here, we developed a mass spectrometry-based method to quantify thymidine dimers in the urine and tested the method on a small group of volunteers after whole-body UVR exposure. Patients and Methods: Years of research resulted in a method based on the “dilute-and-shoot” principle and ultra-performance liquid chromatography (UPLC) coupled to mass spectrometry. The whole body of each of eight healthy volunteers was exposed to 1.5-2.0 standard erythema doses (SEDs) of UVR for 3 consecutive days. Morning urine was collected on Day 1 (before irradiation) and on the following 7-9 days. Prior to analysis, sample preparation consisted of a simple dilution. A tandem quadrupole mass spectrometer coupled to UPLC was used for quantitative analysis in the multiple reaction monitoring mode. Results: After 3 consecutive days of 1.5-2 SEDs, the highest level of thymidine dimer excretion occurred on Day 6 (0.7 ng/ml urine). Compared with baseline, significantly more thymidine dimers were excreted every day until Day 8 (p<0.016). Our method quantifies thymidine dimers that are excreted as dimers (i.e., not degraded further) after nucleotide excision repair. Conclusion: This is the first published mass spectrometry-based method for quantifying thymidine dimers in the urine after whole-body UVR exposure.