PROJECT SUMMARY / ABSTRACT Extracellular vesicles (EVs) exhibit high heterogeneity in biofluids, a feature that traditional bulk-level analysis approaches fail to capture in terms of individual variations. While numerous techniques exist for single-EV analysis, the majority focus primarily on profiling surface proteins. Transcriptional analysis at the level of individual EVs, however, remains largely unexplored. To bridge this gap, we propose the development of a technology for multimodal profiling of individual EVs, leveraging next-generation sequencing and an optimized method for multiplex library preparation. This proposed platform will serve as a unique tool for high-throughput, integrative profiling of single-EV gene expression and surface proteins. It aims to offer high-sensitivity, multi- dimensional biological insights, thereby potentially accelerating the advancement of EV-based diagnostics and targeted therapies.