Tens of millions of women undergo population-based screening for breast cancer by mammography. Despite the life-saving potential of early detection, screening guidelines for mammography are controversial and continue to evolve. At issue are the specificity of mammography for distinguishing benign vs cancerous lesions, the sensitivity of mammography in some patient populations (e.g. women with dense breasts), and concerns regarding over-diagnosis (e.g. some ductal carcinoma in situ). Our ultimate clinical aim is to develop a blood test for early detection of breast cancer that can be used in conjunction with mammography to improve sensitivity and specificity during screening, and thus have enormous clinical impact by detecting treatable cancers missed by mammography (reducing mortality) and by avoiding unnecessary invasive procedures for benign or non-life-threatening disease. Despite considerable effort, attempts to identify blood-based screening biomarkers for early detection of breast cancer have failed, due to technological and methodological limitations. Clearly new approaches are warranted. We are proposing a completely novel strategy, based on the initial discovery of candidate biomarkers in the plasma of “avatar mice” (harboring early passage human breast cancer xenografts), and followed by biomarker triage and validation using a novel biomarker pipeline based on a targeted form of mass spectrometry (MS), multiple reaction monitoring (MRM). If successful, this study could provide a road map for applying this general approach to other cancer sites, beyond breast cancer. Briefly, candidate circulating biomarkers identified in the avatar mouse plasma will be verified in the plasma from the human patients and prioritized for further testing based on integrative proteo-genomic analyses using large breast cancer datasets generated by NCI genomic and proteomic consortia (TCGA and CPTAC). A novel, multiplex MRM-based assay will be developed and analytically validated (according to established fit-forpurpose guidelines) to quantify up to 50-100 prioritized biomarker candidates. Candidate biomarkers will be evaluated in an existing, strongly unbiased collection of plasma samples in which plasmas were collected prior to biopsy, compliant with PRoBE study design criteria, under an SOP from women with undiagnosed mammographic lesions. Performance of the candidate biomarkers will initially be assessed in a training set (100 cases, 100 controls) to verify which candidates show a mean difference in plasma levels between cases vs controls, and to test the possibility of building a multiple marker prediction model. Subsequently, individual candidate biomarkers (as well as a potential multiple marker prediction model) will be assessed in an independent validation set (150 cases, 150 controls) to estimate the sensitivity and specificity of the markers/panel.