Conventional assays such as immunohistochemistry (IHC) and in situ hybridization (ISH) used in clinical procedures for quantification of the human epidermal growth factor receptor-2 (HER2) status in breast cancer have many limitations. In the current study, we have used HER2 expression in a broad range of breast cancer phenotypes to explore the potential utility of a novel immunodetection technique using Raman spectroscopy and Raman imaging combined with artificial intelligence models. The correlations between the Raman method and conventional HER2 testing methodologies (IHC and ISH) have been tested. Raman measurements showed a strong linear correlation (p = 0.05, R2=0,9816) with IHC analysis in the studied breast cell lines: MCF-10A, MCF-7, MDA-MB-231, HTB-30 (SK-BR-3), and AU-565 represent normal, nontumorigenic epithelial cells, triple-positive breast carcinoma, and triple-negative breast cancer cell lines. Analytic testing of Raman spectroscopy and Raman imaging demonstrated that this method may offer advantages over the currently used diagnostic methodologies.