Extracellular vesicles (EVs) have emerged as important intercellular signalling mediators. They carry proteins, nucleic acids and metabolites which can be transferred to a recipient cell, locally or at a distance, to elicit a functional response. The advent of quantitative mass spectrometry-based proteomics, in conjunction with advances in molecular cell biology, and EV purification strategies, has contributed significantly to our improved characterization and understanding of the molecular composition and functionality of EVs. Extracellular vesicle surface proteome (surfaceome) acts as a fundamental signalling gateway by bridging intra- and extracellular signalling networks, dictates EVs’ capacity to communicate and interact with their environment, and is a source of potential disease biomarkers and therapeutic targets. We describe critical insights into diverse proteins operating at the interactive platform of specific types of EVs (including cardiac-derived) and molecular leads for future studies seeking to decipher EV heterogeneity and function.