Abstract Introduction: Breast cancers that express low levels of HER2 that are not considered HER2-positive disease by current HER2 classification standards are the targeted treatment populations for many new and promising HER2-targeted therapies, e.g. HER2 antibody-drug conjugates, tyrosine kinase inhibitors and vaccines. HER2 status is routinely determined by using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). These technologies may have limited utility or lack the sensitivity required to adequately stratify low HER2 protein expression. Following upon the successes of the HERmark Breast Cancer Assay, we have developed the Enhanced Sensitivity HERmark Assay (ESHA) to quantify low HER2 protein expression in formalin-fixed, paraffin-embedded (FFPE) samples. The ESHA format relies on dual-antibody binding for the proximity-dependent release of a fluorescent reporter, which is measured with high sensitivity and reproducibility via capillary electrophoresis to accurately quantify HER2 protein expression. Methods: The HERmark and HERmark ESHA assays utilize HER2 mAb8 conjugated to a light-releasable VeraTag® reporter and HER2 mAb15 conjugated to a photosensitizer. Low HER2 expressing cancer cell lines were used to optimize assay conditions that improved signal strength and extended the lower end of the dynamic range of the HERmark assay. FFPE cancer cell lines and breast tumors were utilized to evaluate assay background, reproducibility, and to compare HER2 assessments based on HERmark, HERmark ESHA and HER2 IHC. Results: The ESHA format expanded the HERmark assay lower limit of quantification by more than 10-fold, and increased the relative fluorescent signal by an average of 16-fold, which also resulted in a decrease in assay variation. ESHA measurements of HER2 protein in a panel of FFPE cell lines correlated with HER2 gene expression levels as reported in the cancer cell line encyclopedia (https://portals.broadinstitute.org/ccle, R2 = 0.857). ESHA measurements of HER2 protein correlated well with conventional IHC scores, along with the added ability to quantitatively distinguish HER2 protein expression levels. Conclusions: We have developed an Enhanced Sensitivity HERmark Assay for the quantification of low HER2 protein expression in formalin-fixed, paraffin-embedded tissue samples. This assay can be a particularly useful tool for the development of novel HER2-targeting therapies in patient populations that are not considered HER2 positive by conventional HER2 classification standards. Improving the stratification of HER2 expression may better define, and perhaps expand, the number of patients that are more likely to benefit from HER2-targeted therapy. Citation Format: Gerald Wallweber, Roy Ravanera, Paul Theobald, Weidong Huang, Christos Petropoulos. Enhanced sensitivity HERmark® assay: A sensitive and quantitative immunoassay superior to immunohistochemistry for the measurement of low HER2 protein levels in formalin-fixed, paraffin-embedded samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1613.