Studies have shown that extracellular vesicles (EVs) are involved in the development of many diseases. Because EVs are released in body fluids, analysis of their protein content can potentially be used as diagnostic and prognostic tools. In this thesis, efficient and simple strategies to improve the EV isolation efficiency with an affinity-based method were developed. Furthermore, optimization of immunoassays towards multiplexed EV protein quantification was performed. Firstly, the optimal magnetic bead sizes for efficient and rapid aptamer-based EV capture were determined. 1 and 2-3 μm aptamer functionalized magnetic beads provided complete and faster isolation of the EV-bead complex (EV bounds to the aptamer functionalized beads) than the nanometer ones (50 and 70 nm). Secondly, blocking reagents on a variety of commonly used substrates for protein measurement in complex biofluids were systematically optimized. It was determined that the choice of blocking strategies highly depends on the substrate. These studies offer strategies for improving EV isolation efficiency and for optimizing EV protein quantification assays, which are crucial for development of non-invasive diagnostic tests for the early detection of diseases.