We describe here a method, termed immunoFISH, for simultaneous in situ analysis of the composition and distribution of proteins and individual RNA transcripts in single cells. Individual RNA molecules are labeled by hybridization and target proteins are concurrently stained using immunofluorescence. Multicolor fluorescence images are acquired and analyzed to determine the abundance, composition, and distribution of hybridized probes and immunofluorescence. We assessed the ability of immunoFISH to simultaneous quantify protein and transcript levels and distribution in cultured HER2 positive breast cancer cells and human breast tumor samples. We demonstrated the utility of this assay in several applications including demonstration of the existence of a layer of normal myoepithelial KRT14 expressing cells that separate HER2+ cancer cells from the stromal and immune microenvironment in HER2+ invasive breast cancer. Our studies show that immunoFISH provides quantitative information about the spatial heterogeneity in transcriptional and proteomic features that exist between and within cells.