The objective of this study was to explore the characteristics and roles of extracellular vesicles (EVs) obtained from human mesenchymal stem cells (MSCs) under two culture conditions: undifferentiated MSCs (Naïve-EVs) and MSCs undergoing early-stage osteogenesis (Osteo-EVs). Additionally, the study aimed to evaluate the potential of Osteo-EVs to induce bone formation for the purpose of regenerating bone defects in rat calvaria. To accomplish these goals, EVs from both groups were isolated using a technique called size-exclusion chromatography. The isolated EVs were then subjected to various analyses to gain insights into their properties. This included examining their size distribution, morphology, flow cytometry analysis, and proteome profiling using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with Label Free Quantification.