UV-induced formation of photoproducts in DNA is a major initiating event of skin cancer. Consequently, many analytical tools have been developed for their quantification in nuclear DNA. In the present work, we developed an assay for the quantification of the pyrimidine dimers in easily accessible biological fluids like urine for in vivo studies and cell culture medium for in vitro investigations. We targeted the DNA photoproducts present in the short oligonucleotides released by the nucleotide excision repair machinery. Unlike other approaches, our assay does not only quantify the dinucleotides expected to be released from these repair products by action of endogenous nucleases. Our technique combines isolation of the photoproducts-bearing oligonucleotides to enzymatic hydrolysis and HPLC-tandem mass spectrometry detection with isotopic dilution. Biological validation of the approach was first obtained from experiments in HaCat keratinocytes. We could unambiguously follow the transfer of photoproducts first from nuclear DNA to cytoplasm, and in a second step from cytoplasm to culture medium. Release of pyrimidine dimers in the culture medium was also observed in dose- and time-dependent manners from reconstructed human epidermis and human skin explants. Relevance of the assay to human exposure was shown by the detection of pyrimidine dimers in the urine of volunteers collected after recreational exposure in summer. Experiments in RHE and a first clinical study showed that detection of DNA photoproducts in fluids could be a valuable tool in the assessment of photoprotection. Reference Reynaud, N., Belz, L., Beal, D., Bacqueville, D., Duplan, H., Genies, C., Questel, E., Josse, G. and Douki, T. (2022) DNA photoproducts released by repair in biological fluids as biomarkers of the genotoxicity of UV radiation. Anal. Bioanal. Chem. 414, 7705-7720. DOI: 10.1007/s00216-022-04302-1