Human epidermal growth factor receptor 2 (HER2) protein is often overexpressed in breast cancer and is correlated with a worse prognosis and thus accurate detection of HER2 is crucial to provide appropriate cares for patients. However, none of the techniques are the universal gold standard to detect accurate HER2 status. In this context, we established a multiple reaction monitoring-mass spectrometry (MRM-MS) assay that improves upon the conventional methods for differentiating HER2 status in FFPE tissues. We quantified the HER2 peptides in 210 FFPE tissues including HER2 0 (n=30), 1+ (n=30), 2+FISH- (n=61), 2+FISH+ (n=59), and 3+ (n=30). We applied the ratio between the quantitative data of HER2 peptides and normalization factors as a specific value for determining HER2 status to raise the accuracy of the quantification of HER2. In order to determine whether the data generated by MRM assay matched with the data obtained by IHC and FISH scores, the quantitative data of a HER2 peptide normalized by a quantitative data of Junctional adhesion molecule A (JAM-1) peptide were used. The Mann Whitney U test determined that significant differences were found in all the HER2 and FISH groups, and especially the MRM data can distinguish between HER2 2+FISH- and HER2 2+FISH+ (p<0.000), which cannot be differentiated by IHC. In addition, the MRM data distinguished the HER2-negative group and the HER2-positive group that is expected to benefit from trastuzumab therapy (p<0.000). The novel MRM assay that we developed clearly distinguished the equivocal HER2 status that cannot be classified by the conventional method, IHC, as well as the overall HER2 classification. Our developed assay using MRM for determining HER2 status would provide clinicians with valuable diagnostic information and ensure that all patients who have HER2-positive breast cancer have the opportunity to receive proper treatment.