Exosomes (small extracellular vesicles) in living organisms play an important role in processes such as cell proliferation or intercellular communication. Recently, exosomes have been extensively investigated for biomarker discoveries for various diseases. An important aspect of exosome analysis involves the development of enrichment methods that have been introduced for successful isolation of exosomes. These methods include ultracentrifugation, size exclusion chromatography, polyethylene glycol-based precipitation, immunoaffinity-based enrichment, ultrafiltration, and asymmetric flow field-flow fractionation among others. To confirm the presence of exosomes, various characterization methods have been utilized such as Western blot analysis, atomic force microscopy, electron microscopy, optical methods, zeta potential, visual inspection, and mass spectrometry. Recent advances in high-resolution separations, high-performance mass spectrometry and comprehensive proteome databases have all contributed to the successful analysis of exosomes from patient samples. Herein we review various exosome enrichment methods, characterization methods, and recent trends of exosome investigations using mass spectrometry-based approaches for biomarker discovery.