Abstract There is widespread interest in the relationship between aging and cancer, and the involvement of senescence in both processes. The accumulation of senescent cells imparts elevated risk of age-associated disease, including cancer; however, a continuing challenge in the field is the lack of senescence-specific biomarkers. Chemotherapy and radiotherapy are potent inducers of senescence, and the pro-inflammatory secretion of persistent senescent cells has been shown to propel tumor evolution and metastatic dissemination. Thus, there is a need to define how much senescence can be tolerated in tissue prior to physiologic dysfunction; however defining senescence ‘burden’ in aged tissue or cancer cannot be accurately determined with existing markers due to their redundancy. To address this shortcoming, we undertook a proteomic-based analysis to identify novel senescent biomarkers in K-Ras mutant non-small cell lung cancer (NSCLC). Importantly, quiescent cells were also included in the experimental design to enable the identification of proteins associated with general dormancy and not specific to senescence. Cell surface proteins from senescent, quiescent and asynchronous cancer cells were labeled with biotin, enriched and analyzed using tandem mass spectrometry. An average of 850 proteins were detected in each replicate, and 287 were unique to senescence. Gene ontology analysis of proteins detected in senescence indicated functions enriched for metabolism, DNA damage checkpoint integrity, membrane organization, wound healing and cytoskeletal remodeling; all consistent with a senescent phenotype. Data were filtered by strict criteria to derive a final list of 31 proteins unique to senescence. Initial validation of a subset of these for which specific commercial antibodies were available, was done by immunoblotting whole cell lysates and plasma membrane-enriched fractions. Some candidates had enriched expression in senescence; while others were increased in both senescence and quiescence and thus are more useful as dormancy markers. Additional validation by immunofluorescence was performed to deduce expression and localization of four senescent candidates. Novel patterns of localization were found whereby some candidates showed enrichment in structures resembling large vesicles and invadopodia-like extensions that connected with neighboring senescent cells. Lastly, immunohistochemical (IHC) analyses of formalin-fixed tissue obtained from chemotherapy-treated NSCLC patients revealed intense staining in enlarged cells with polymorphic nuclei, a characteristic of senescent cells. Thus, we have identified a unique cohort of senescence-specific biomarkers, some of which are amenable to IHC analyses. Ongoing studies will determine their utility as prognostic and predictive biomarkers of senescence tumor burden in NSCLC, with potential extrapolation to other malignancies and pathologies. Citation Format: Meagan Vogt, Chia-Ping H. Yang, Edward Nieves, Kenny Ye, Perry Cohen, Steven Keller, Hayley M. McDaid. Proteomic-based senescent biomarker identification and characterization in non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-066.