Abstract: Human erythrocytes are an excellent model system to study interaction of pro-oxidants with membranes. The present study, was carried out to evaluate the in vitro antioxidant properties and protective effects of silymarin (milk thistle) in human erythrocyte against tert-butyl hydroperoxide (t-BHP), a potent oxidative stress inducer chemical. Protective effect of silymarin was assessed in vitro by monitoring the antioxidant marker glutathione (GSH) and malondialdehyde (MDA) in three groups of erythrocytes: (I) vehicle control, (II) tert-butyl hydroperoxide incubated group and (III) t-BHP co-incubated with various concentrations of silymarin. The effects of silymarin on lipid peroxidation MDA and GSH content in erythrocytes were assessed. It was observed that activity of GSH was significantly decreased and the malondialdehyde levels were elevated when human erythrocytes were incubated with tert-butyl hydroperoxide in concentration as well as time dependent manner. The protective effect of silymarin is elucidated by the significant reversal of the GSH content and malondialdehyde reduction in human erythrocyte. Therefore the results of our present study suggested that silymarin possess substantial protective effect against in-vitro induced oxidative stress damages by t-BHP.