Peroxynitrite (ONOO-) is a reactive oxidant formed from superoxide (*O2(-)) and nitric oxide (*NO), that can oxidize several cellular components, including essential protein, non-protein thiols, DNA, low-density lipoproteins (LDL), and membrane phospholipids. ONOO- has contributed to the pathogenesis of diseases such as stroke, heart disease, Alzheimer's disease, and atherosclerosis. Because of the lack of endogenous enzymes to thwart ONOO- activation, developing a specific ONOO- scavenger is remarkably important. In this study, the ability of hesperetin (3',5,7-trihydroxy-4-methoxyflavanone) to scavenge ONOO- and to protect cells against ONOO- and ROS was investigated. The data gained show that hesperetin can efficiently scavenge authentic ONOO- . In spectrophotometric analysis, the data revealed that hesperetin led to declined ONOO- -mediated nitration of tyrosine through electron donation. Hesperetin exhibited significant inhibition on the nitration of bovine serum albumin (BSA) by ONOO- in a dose-dependent manner. Hesperetin also manifested cytoprotection from cell damage induced by ONOO- and ROS. The present study suggests that hesperetin is a powerful ONOO- scavenger and promotes cellular defense activity in the protection against ONOO- involved diseases.