Accumulated evidence points to DNA repair capacity as an important factor in cancer and other diseases. DNA repair proteins are promising drug targets and are emerging as prognostic and therapeutic biomarkers. Thus, the knowledge of the overexpression or underexpression levels of DNA repair proteins in tissues will be of fundamental importance. In this work, mass spectrometric assays were developed for the measurement in tissues of the human DNA repair protein NEIL1 (hNEIL1), which is involved in base excision and nucleotide excision repair pathways of oxidatively induced DNA damage. Liquid chromatography/isotope-dilution tandem mass spectrometry (LC-MS/MS), in combination with a purified and fully characterized recombinant (15)N-labeled analogue of hNEIL1 ((15)N-hNEIL1) as an internal standard, was utilized to develop an accurate method for the quantification of hNEIL1. Both hNEIL1 and (15)N-hNEIL1 were hydrolyzed with trypsin, and 18 tryptic peptides from each protein were identified by LC-MS/MS on the basis of their full-scan mass spectra. These peptides matched the theoretical peptides expected from trypsin hydrolysis of hNEIL1 and provided a statistically significant protein score that would unequivocally identify hNEIL1. The product ion spectra of the tryptic peptides from both proteins were recorded, and the characteristic product ions were defined. Selected-reaction monitoring was used to analyze mixtures of hNEIL1 and (15)N-hNEIL1 on the basis of product ions. Additional confirmation of positive identification was demonstrated via separation of the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in-gel tryptic digestion followed by LC-MS/MS analysis. These results suggest that the developed assays would be highly suitable for the in vivo positive identification and accurate quantification of hNEIL1 in tissues.