Objective The vascular endothelium provides a unique interaction plane for plasma proteins and leukocytes in inflammation. It has been established that particular proteins, including ICAM1, VCAM1 and SELE, are upregulated on the endothelial cell surface in the presence of pro-inflammatory cytokines Tumor Necrosis Factor α (TNFα) and Interleukin 1β (IL-1β). We now map cytokine-induced proteomic alterations to gain further insight into endothelial inflammation. Approach and results We evaluated alterations in the in-depth proteome, cell surface proteome and phosphoproteome after 24 hours of cytokine stimulation. In addition, protein expression profiles were measured after 4, 8, 16 and 24 hours of stimulation. We found that cytokine-stimulation induced a two-step response. After 4 hours, initial protein alterations were detected, including upregulation of ICAM1 and SELE, followed by a second burst of regulation after 8 to 16 hours, which included proteins involved in antigen processing, cytokine production, virus defense and ECM-interaction. Furthermore, we found that TNFα and IL-1β provoke a highly similar endothelial response with few specific alterations. Using a novel cell surface proteomics approach, we observed highly similar changes on the cell surface. Two surface proteins displayed altered labeling of specific epitopes, suggesting that these sites are modified or involved in interactions. Combined, our integrated proteomic data describes a full array of affected processes: leukocyte interaction, self-antigen presentation, cytokine production, nitric oxide production and extracellular matrix interaction. Conclusions Our study provides a detailed quantitative proteomic analysis of endothelial inflammation in which novel inflammation-responsive proteins were uncovered, and a two-step cytokine response was identified.