Background: Conventional HER2-targeting therapies improve outcomes for patients with HER2-positive breast cancer (BC), defined as tumors showing HER2 protein overexpression by an immunohistochemistry (IHC) assay and/or ERBB2 gene amplification determined by in situ hybridization (ISH). Emerging HER2-targeting compounds show benefit in some patients with neither HER2 protein overexpression nor ERBB2 gene amplification, creating a need for new assays to select HER2-low tumors for treatment with these compounds. Here, we report the analytical performance of a targeted mass spectrometry-based assay for quantifying HER2 protein in formalin fixed paraffin-embedded (FFPE) and frozen BC biopsies. Methods: We used immuno-enrichment coupled to multiple reaction monitoring-mass spectrometry (immuno-MRM-MS) to quantify HER2 protein (as peptide GLQSLPTHDPSPLQR) in 96 frozen and 119 FFPE BC biopsies. We characterized linearity, lower limit of quantification (LLOQ), and intra- and inter-day variation of the assay in frozen and FFPE tissue matrices. We determined concordance between HER2 immuno-MRM and predicate IHC and ISH assays and examined the benefit of multiplexing the assay to include proteins expressed in tumor sub-compartments (e.g. stroma, adipose, lymphocytes, epithelium) to account for tissue heterogeneity. Results: HER2 immuno-MRM assay linearity was ≥103, assay coefficient of variation (CV) was 7.8% (FFPE) and 5.9% (frozen), and endogenous measurement CV was 7.7% (FFPE) and 7.9% (frozen). Immuno-MRM-based HER2 measurements strongly correlated with predicate assay HER2 determinations, and concordance was improved by normalizing to GAPDH. HER2 was quantified above LLOQ in all HER2-low and -negative tumors. Conclusions: Immuno-MRM-MS can be used to quantify HER2 in FFPE and frozen BC biopsies, even at low HER2 expression levels.