Ultraviolet radiation from sunlight causes DNA damage in skin cells by formation of photoproducts, mainly cyclobutane pyrimidine dimers (CPD), which are reverted by exogenous CPD-photolyase, preventing photoaging and skin cancer. High performance liquid chromatography tandem mass spectrometry method for quantification of CPD-photolyase activity was developed to search new enzymes sources for dermatology or clinical studies. The method was based in the enzymatic conversion of a 15mer oligonucleotide, containing a center cyclobutane thymidine dimer, to the restored 15mer oligonucleotide. Three ion pair reagent were evaluated by response surface methodology to increase mass intensities. Additionally, chromatographic separation of oligonucleotides was performed. The selected mobile phase was 15 mM diisopropylethylamine/20 mM hexafluoroisopropanol in methanol. The method allowed total separation between the oligonucleotides studied (resolution of 2.3) by using the core shell technology, which reduce the diffusion time of the analyte into the column, increasing the efficiency and minimizing the analysis time at 7 min. The mass spectrometry detection allowed a high selectivity and sensitivity. This is the first time where MRM modality has been employed with this specific purpose. Oligonucleotides recovery from reaction mixture was ∼ 94% and the limit of quantification was 13.4 nM for 15mer. The method was evaluated with a recombinant CPD-photolyase from Synechococcus leopoliensis using purified and crude protein extract. CPD-photolyase could be measured in terms of activity for enzymatic kinetics studies, for evaluation of UV-R effects in (micro)organisms and to identify new enzymes.