Exosomes are cell-derived small extracellular membrane vesicles (50–100 nm in diameter) actively secreted by a number of healthy and diseased cell types. Exosomes can mediate cellular, tissue, and organ level micro communication under normal and pathological conditions by shuttling proteins, mRNA, and microRNAs. Prior to vesicle molecular profiling, these exosomes can be isolated from conditioned cell media or bodily fluids such as urine and plasma in order to explore the contents and functional relevance. Exosome purification and analyses are a fast-growing research field. Regardless of several advances in exosome purification and analyses methods, research still faces several challenges. Despite tremendous interest in the role of extracellular vesicles, there is no general agreement on dependable isolation protocols. Therefore, there is an urgent need to establish reliable protocol of exosome purification and analysis. Here, we report a simple cost-effective isolation and analysis of cardiac myocyte exosomes from conditioned media.