A critical factor to favor a good prognosis in breast cancer (BC) patients is early detection. Offering prompt diagnosis, treatment, and delaying or stopping the progression of the disease are the key aspects of breast cancer management. The use of FTIR spectroscopy on ex vivo breast samples can elucidate important biochemical information associated with the presence and progression of breast cancer. In this study, tissue microarrays of breast cancer biopsy samples (n = 378), and normal breast (n = 134) were analyzed using FTIR spectroscopy, principal component analysis (PCA) for feature extraction, and validation employing linear discriminant analysis (LDA). The differentiation between normal breast and breast cancer was successfully achieved with a sensitivity of 92% and specificity of 86%. Lipids and proteins spectral bands were identified as the main differentiators between normal and breast cancer. FTIR results also highlighted that chemical structural changes formed an important part of breast cancer evolution. Regardless of the heterogeneity of breast cancer, the use of multivariate analysis and FTIR spectroscopy offers a suitable and reliable tool for BC monitoring and diagnosis. The integration of spectroscopic techniques such as FTIR in cancer diagnosis and monitoring could add useful information enabling the diagnosis and management of breast cancer patients.