Exosome isolation and analysis play a pivotal role in unlocking the secrets of intercellular communication and biomarker discovery. Exosomes, small extracellular vesicles secreted by various cell types, are rich in bioactive molecules such as proteins, nucleic acids, and lipids. Isolating exosomes from biological samples requires specialized techniques that separate them from other components such as cells, debris, and proteins. Once isolated, exosomes can undergo a myriad of analyses to elucidate their contents and functions. Proteomic analysis enables the identification and quantification of proteins within exosomes, shedding light on their cellular origin and physiological roles. Genomic and transcriptomic analyses provide insights into the nucleic acid cargo of exosomes, including DNA, RNA, and non-coding RNAs, which may serve as potential biomarkers for various diseases. Additionally, lipidomic analyses reveal the lipid composition of exosomes, offering clues to their biogenesis and intercellular signaling functions. Integrating these diverse analytical approaches allows for a comprehensive understanding of exosome biology and opens avenues for their diagnostic and therapeutic applications in various fields, including cancer, neurodegenerative diseases, and regenerative medicine.