Shock induces oxidative stress by ischemia-reperfusion phenomenon. Endothelial cells are involved in the inflammatory response and oxidative stress responsible for microcirculation impairment and organ failure. We examined the potential of serum from patients to induce in vitro reactive oxygen species production by cultured human umbilical vein endothelial cells (HUVECs). Three groups were compared: hemorrhagic shock trauma patients, isolated brain injured patients, and healthy volunteers. In the hemorrhagic shock group we sought a correlation between reactive oxygen species production and severity of shock. Serum was separated and perfused in an in vitro model of perfused HUVECs. Ex vivo reactive oxygen species production was assessed by fluorescence microscopy using dichlorodihydrofluorescein, an intracellular dye oxidized by H2O2. Results are expressed in proportional change from baseline and normalized by protidemia to control for variation related to hemodilution. Reactive oxygen species production by endothelial cells exposed to serum from hemorrhagic shock patients (46.2±24.9%) was significantly greater than in those with brain injury (3.9±35.1%) and in healthy volunteers (−6.8±5.8%). In the hemorrhagic shock group dichlorodihydrofluorescein fluorescence was strongly correlated positively to Simplified Acute Physiology Score II and lactatemia and negatively to [HCO3−]. Serum from trauma patients with hemorrhagic shock induces reactive oxygen species formation in naive endothelial cells which is correlated to shock severity.