Therapies like ipilimumab and nivolumab have shown the potential for approaches that direct the patient’s own immune system against tumors. Further advances will require a detailed understanding of the tumor microenvironment and characterization of the location and status of immune cells and their interaction with tumor cells. This will require methods that provide phenotyping of immune and cancer cells combined with the cytoarchitectonics of the tumor. To achieve this, a practical method for simultaneous immunohistochemistry of up to 8 biomarkers in a single tissue section using standard unlabeled primary antibodies will be described. In addition, approaches for multiplexed imaging, single cell quantitative analysis, automated phenotyping and bioinformatics that enable new insights into cancer biology and immunology will be presented. These will then be leveraged in analyses of multiple cancer types, where it is established that the host-tumor interaction is complex and difficult to characterize with standard immunohistochemistry or flow cytometry. But by leveraging multiplexed IHC in situ, we will demonstrate the unique spatial relationships of immune phenotypes in and around both epithelial and non-epithelial tumor types, and show how this data has the potential to form the basis of assays that can guide therapy and monitor response. Further data regarding functional assessment of regulatory immunologic co-factor expression will be discussed.