Extracellular vesicles (EVs) are critical mediators of intercellular communication, capable of regulating the transcriptional landscape of target cells through horizontal transmission of biological information, such as proteins, lipids, and RNA species. This capability highlights their potential as novel targets for disease intervention. Areas covered: This review focuses on the emerging importance of discovery proteomics (high-throughput, unbiased quantitative protein identification) and targeted proteomics (hypothesis-driven quantitative protein subset analysis) mass spectrometry (MS)-based strategies in EV biology, especially exosomes and shed microvesicles. Expert commentary: Recent advances in MS hardware, workflows, and informatics provide comprehensive, quantitative protein profiling of EVs and EV-treated target cells. This information is seminal to understanding the role of EV subtypes in cellular crosstalk, especially when integrated with other 'omics disciplines, such as RNA analysis (e.g., mRNA, ncRNA). Moreover, high-throughput MS-based proteomics promises to provide new avenues in identifying novel markers for detection, monitoring, and therapeutic intervention of disease.