Extracellular vehicles such as exosomes which lie in the size range of 30–150 nm have gained significant attention due to their ability to contain molecular cargoes. These vesicles are released by various cells and are found to stably carry their molecular cargoes to recipient cells via different biological fluids. Therefore, exosomes isolated from various fluids such as plasma, urine, and pericardial fluid have been extensively studied in recent years. However, due to the miniscule nature of exosomes, isolation of exosomes from biological fluids has been a challenge, mainly due to low yields, contamination from large vesicles and proteins, and variable starting sample volumes. The following protocol describes the isolation of exosomes from human pericardial fluid samples collected from patients undergoing coronary artery bypass graft surgery. The exosomes isolated from these samples have been previously shown to have increased expression of cardiovascular molecular markers. Due to the precious nature of the patient samples, the exosomes isolated from this protocol have been validated to confirm their purity and viability for downstream applications.