Extracellular vesicles (EVs), particularly exosomes, are increasingly recognized as important mediators of intercellular communication due to their ability to transport a diverse array of bioactive molecules. These vesicles play crucial roles in various physiological and pathological processes and hold significant promise as disease biomarkers, therapeutic agents, and drug delivery vehicles. Exosomes contain specific groups of membrane proteins, lipids, nucleic acids, cytosolic proteins, and other signaling molecules, which dictate their targeting specificity and functional roles upon reaching recipient cells. Despite growing understanding, the molecular mechanisms governing the selective sorting and packaging of cargo within exosomes remain largely unelucidated. This review aims to summarize current insights into the molecular mechanisms that regulate the sorting of various molecules into exosomes, their resulting biological functions, and potential clinical applications, with a focus on their relevance in cancer and other diseases. Understanding the loading processes and mechanisms involved in exosome cargo sorting is essential for uncovering the physiological and pathological roles of exosomes, identifying therapeutic targets, and advancing the clinical development of exosome-based therapeutics. Exosome biogenesis is a complex, highly regulated process involving several sequential stages, from the initial formation of early endosomes to the release of mature exosomes into the extracellular environment. The selective sorting of cargo molecules into nascent exosome vesicles is a critical determinant of exosome functionality, diversity, and specificity. This review explores the various classes of molecules that constitute exosome cargo, the regulatory factors and machinery that govern cargo selection and packaging, and the implications of cargo sorting in disease contexts. Recent advancements in genetic tools and exosome characterization techniques have begun to unveil the intricate mechanisms underlying cargo sorting into exosomes. Exosome engineering strategies, including active and passive approaches, aim to enhance cargo loading and exosomal targeting. Active strategies involve integrating target substances during exosomal biogenesis, while passive approaches involve loading exogenous substances after exosome secretion. These methods have been used to bioengineer exosomes for delivering therapeutic cargo, such as CRISPR/Cas9, superrepressors, and lysosomal enzymes. Overall, the review highlights the importance of understanding exosome cargo sorting mechanisms for advancing the development of exosome-based therapies and their potential applications in disease management.Extracellular vesicles (EVs), particularly exosomes, are increasingly recognized as important mediators of intercellular communication due to their ability to transport a diverse array of bioactive molecules. These vesicles play crucial roles in various physiological and pathological processes and hold significant promise as disease biomarkers, therapeutic agents, and drug delivery vehicles. Exosomes contain specific groups of membrane proteins, lipids, nucleic acids, cytosolic proteins, and other signaling molecules, which dictate their targeting specificity and functional roles upon reaching recipient cells. Despite growing understanding, the molecular mechanisms governing the selective sorting and packaging of cargo within exosomes remain largely unelucidated. This review aims to summarize current insights into the molecular mechanisms that regulate the sorting of various molecules into exosomes, their resulting biological functions, and potential clinical applications, with a focus on their relevance in cancer and other diseases. Understanding the loading processes and mechanisms involved in exosome cargo sorting is essential for uncovering the physiological and pathological roles of exosomes, identifying therapeutic targets, and advancing the clinical development of exosome-based therapeutics. Exosome biogenesis is a complex, highly regulated process involving several sequential stages, from the initial formation of early endosomes to the release of mature exosomes into the extracellular environment. The selective sorting of cargo molecules into nascent exosome vesicles is a critical determinant of exosome functionality, diversity, and specificity. This review explores the various classes of molecules that constitute exosome cargo, the regulatory factors and machinery that govern cargo selection and packaging, and the implications of cargo sorting in disease contexts. Recent advancements in genetic tools and exosome characterization techniques have begun to unveil the intricate mechanisms underlying cargo sorting into exosomes. Exosome engineering strategies, including active and passive approaches, aim to enhance cargo loading and exosomal targeting. Active strategies involve integrating target substances during exosomal biogenesis, while passive approaches involve loading exogenous substances after exosome secretion. These methods have been used to bioengineer exosomes for delivering therapeutic cargo, such as CRISPR/Cas9, superrepressors, and lysosomal enzymes. Overall, the review highlights the importance of understanding exosome cargo sorting mechanisms for advancing the development of exosome-based therapies and their potential applications in disease management.