Here, we developed and validated a robust protocol for smFISH in FFPE breast cancer tissue sections (FFPE-smFISH), and applied it to quantify transcriptional ITH and to perform spatial analysis, at single-cell level, of the two most clinically relevant breast cancer biomarkers: epidermal growth factor receptor 2 (HER2) and estrogen receptor 1 (ER). Here, ESR1 is linked to breast cancer.