Olea europaea L. (Oleaceae) leaves have been used for centuries in folk medicine to treat many degenerative and inflammatory diseases including hypertension, atherosclerosis and cardiovascular disorders. The present study was aimed to quantify the phenolic compounds present in three different extracts of O. europaea leaves, and to demonstrate their protective effects on human umbilical vein endothelial cells against oxidative injury induced by H2O2. Oleuropein, hydroxytyrosol, quercetin, luteolin and rutin in the olive leaf extracts were quantified by using a validated HPLC-UV method. Total phenolic content was determined using the Folin-Ciocalteau assay. The MTT and dichlorofluorescein assays were conducted to measure cytotoxicity and intracellular reactive oxygen species generation, respectively. Oleuropein was the major phenolic component. Each phenolic compound (1.0 and 10.0 mu M) and each extract (10.0 mu g/mL) significantly (p<0.05) preserved human umbilical vein endothelial cells against H2O2-induced toxicity, but olive leaf extract-1 and luteolin seemed to be more effective in studied concentrations even in the inhibition of cellular reactive oxygen species generation (p<0.05). In addition, a positive correlation was found between reactive oxygen species inhibitory activity and total phenol content versus viability protecting capacity of each olive leaf extracts. These results supply a new validated HPLC method for the effective characterization of olive leaf polyphenolic compounds and support the notion that the polyphenolic components of olive leaves are the efficient cytoprotective agents against H2O2-induced oxidative stress and toxicity in human umbilical vein endothelial cells.