A new workflow for protein-based tumor heterogeneity probing in tissues is here presented. Tumor heterogeneity is believed to be key for therapy failure and differences in prognosis in cancer patients. Comprehending tumor heterogeneity, especially at the protein level, is critical for tracking tumor evolution, and showing the presence of different phenotypical variants and their location with respect to tissue architecture. Although a variety of techniques is available for quantifying protein expression, the heterogeneity observed in the tissue is rarely addressed. The proposed method is validated in breast cancer fresh-frozen tissues derived from five patients. Protein expression is quantified on the tissue regions of interest (ROI) with a resolution of up to 100 μm in diameter. High heterogeneity values across the analyzed patients in proteins such as cytokeratin 7, β-actin and epidermal growth factor receptor (EGFR) using a Shannon entropy analysis are observed. Additionally, ROIs are clustered according to their expression levels, showing their location in the tissue section, and highlighting that similar phenotypical variants are not always located in neighboring regions. Interestingly, a patient with a phenotype related to increased aggressiveness of the tumor presents a unique protein expression pattern. In summary, a workflow for the localized extraction and protein analysis of regions of interest from frozen tissues, enabling the evaluation of tumor heterogeneity at the protein level is presented.