Lab head information updated Although the determination of HER2 expression in breast cancer would be of critical importance in the clinical management related to the selection of the patients who are expected to benefit from trastuzumab therapy, the conventional techniques have inherent limitations that disqualify their use as gold standards. Specifically, fluorescence in situ hybridization (FISH), which is mandatory for determining IHC 2+ cases, is time-consuming and economically inefficient. To address this shortcoming, we aimed to discriminate the equivocal HER2 status, which cannot be classified by the conventional method such as IHC, using a highly sensitive and quantitative MRM-MS assay. In order to assess the agreement between MRM-MS and IHC/FISH data, the quantitative data of a HER2 peptide was normalized by junctional adhesion molecule A (JAM1) levels, which is an epithelial cell-specific protein. The normalized HER2 levels differentiated the ambiguous IHC results of. In addition, the MRM-MS data distinguished HER2-negative breast cancer from the HER2-positive breast cancer that was expected to benefit from trastuzumab therapy. The newly developed HER2-targeted proteomic assay may provide a general strategy for complementing the limitations of conventional HER2-targeted therapeutic strategies.