Endothelial microparticles (EMPs) are small vesicles released from endothelial cells (ECs) and found circulating in the blood. EMPs are formed by a plasma membrane surrounding a small amount of cytosol and contain a subset of cellular proteins. As the number of EMPs in the blood increases with certain diseases, they may be an attractive biomarker for clinical diagnosis. Proteomic analysis of EMPs has been previously performed by mass spectrometry. However, the proteomic information of the ECs that secrete EMPs is lacking. This study introduces an in vitro model of activated ECs we created for proteomic analyses and reports the changes of the protein content in the ECs and EMPs using proteomic methods. Thus, this study provides valuable information for the analysis of the highly dynamic secretion process of EMPs. There is a direct correlation between the proteins that form EMPs and tumor necrosis factor-α (TNF-α)-activated ECs. The endothelial proteins transferred by EMPs may play important roles in the interaction between EMPs and the target cells, which may lead to endothelial dysfunction.