Cells live in a close social context by having mutual communication with their local microenvironment. This complex intercellular communication activates dynamic signaling pathways and regulates specific cell fate. MS-based proteomics has been approved to be inevitable for characterizing dynamic protein expression and PTMs on a global scale. However, because of technical difficulties for targeting membrane receptors and secreted proteins, especially in a physiologically relevant manner, systematic characterization of intercellular signaling by MS-based proteomics has largely lagged behind. Here, I will review the latest proteomics technology development and its application to characterizing different modes of intercellular communication including indirect and direct cell–cell communication, and protein translocalization. I will discuss how MS-based proteomics has been applied for systems-level profiling intercellular signaling in defined biological contexts including tumor microenvironment, bacteria/virus–host cell interaction, immune cell interaction, and stem cell niche.