Increasing evidence indicates the importance of the tumor microenvironment, in particular cancer associated fibroblasts, in cancer development and progression. In this study we developed a novel, visually based method to identify new immunohistochemical signatures of these fibroblasts. The method employed a protein list based on 759 protein products of genes identified by RNA profiling from our previous study, comparing fibroblasts with differential growth modulating effect on human cancers cells, and their first neighbors in the human protein interactome. These 2,654 proteins were analyzed in the Human Protein Atlas online database by comparing their immunohistochemical expression patterns in normal vs. tumor associated fibroblasts. Twelve new proteins differentially expressed in cancer associated fibroblasts were identified (DLG1, BHLHE40, ROCK2, RAB31, AZI2, PKM2, ARHGAP31, ARHGAP26, ITCH, EGLN1, RNF19A, PLOD2), four of them can be connected to the Rho kinase signaling pathway. They were further analyzed in several additional tumor stromata and revealed that the majority showed congruence amongst the different tumors. Many of them were also positive in normal myofibroblast like cells. The new signatures can be useful in immunohistochemical analysis of different tumor stromata and may also give us an insight into the pathways activated in them in their true in vivo context. The method itself could be used for other similar analysis to identify proteins expressed in other cell types in tumors and their surrounding microenvironment. © 2013 Wiley Periodicals, Inc.