The extracellular vesicle (EV) surface proteome (surfaceome) acts as a fundamental signalling gateway by bridging intra-and extracellular signalling networks, dictates EVs’ capacity to communicate and interact with its the/their environment, and is a source of potential disease biomarkers and therapeutic targets. However, our understanding of surface protein composition of large EVs (100-1000 nm), a major EV-subtype remains limited. Our study provides critical insights into proteins operating at the large interactive platform of L-EVs, and molecular leads for future studies seeking to decipher relatively-understudied L-EV form, heterogeneity and function.