Exosomes are small membrane vesicles released by the fusion of multivesicular bodies (MVBs) with the plasma membrane. Initially considered as cellular waste, exosomes have been increasingly recognized for their roles in cell-to-cell communication and their potential in therapeutic and diagnostic applications. The formation and release of exosomes involve several cellular processes, including the formation of intraluminal vesicles (ILVs) within MVBs, transport of MVBs to the plasma membrane, and fusion of MVBs with the plasma membrane. Key molecules involved in these processes include the endosomal sorting complex required for transport (ESCRT) proteins, tetraspanins, and lipid-modifying enzymes. The composition of exosomes is influenced by both protein and lipid molecules, and their release can be regulated by cellular conditions such as stress and autophagy. Methodological challenges in exosome isolation and quantification, such as the use of different centrifugation methods and the presence of contaminants, can affect the reproducibility of studies. Understanding the molecular mechanisms behind exosome biogenesis and release is crucial for their clinical application and further exploration of their roles in health and disease.Exosomes are small membrane vesicles released by the fusion of multivesicular bodies (MVBs) with the plasma membrane. Initially considered as cellular waste, exosomes have been increasingly recognized for their roles in cell-to-cell communication and their potential in therapeutic and diagnostic applications. The formation and release of exosomes involve several cellular processes, including the formation of intraluminal vesicles (ILVs) within MVBs, transport of MVBs to the plasma membrane, and fusion of MVBs with the plasma membrane. Key molecules involved in these processes include the endosomal sorting complex required for transport (ESCRT) proteins, tetraspanins, and lipid-modifying enzymes. The composition of exosomes is influenced by both protein and lipid molecules, and their release can be regulated by cellular conditions such as stress and autophagy. Methodological challenges in exosome isolation and quantification, such as the use of different centrifugation methods and the presence of contaminants, can affect the reproducibility of studies. Understanding the molecular mechanisms behind exosome biogenesis and release is crucial for their clinical application and further exploration of their roles in health and disease.