Tramadol   is a widely used analgesic that stimulates the μ opioid receptor and inhibits serotonin and noradrenalin reuptake. In this study, we investigate the effect of chronic administration of 200 and 400mg of tramal for 15,30 ,45 days followed by withdrawal periods(w15) on protein activity in male rats as manifested by changes in electrophoretic serum protein patterns  and gene expression manifested by DNA damage, measured  by Comet Assay .   Materials and methods: 100 male Wistar rats (100-150 g) were included and divided into three groups, control group (n = 20), Tramal(I) group (n = 40), received the drug orally at doses of 200mg/kg/day for 15,30,45 days of the study, respectively(10 rats for each subgroup).TR45 group was followed by 15 withdrawal  period(W15) ,Tramal(II) group (n = 40), received the drug orally at doses of400mg/kg/day for 15,30,45 days of the study, respectively. TR45 group was followed by 15 withdrawal period (W15).    Results:   Results exhibited major changes in the protein pattern which included changes in the molecular weight of the control bands and the relative percentage of protein fraction as well as the total number of bands, as a result of disappearance of some original bands and appearance of other new one. Serum protein fraction revealed an increase in total number of protein fractions being in Tr200mg (14 and 16 bands), whileTR400mg revealed a decrease in total number of bands (except TR15 which exhibited an increase in bands (14). The changes were observed all over the treated   groups as well as in the withdrawal groups In this study, the alkaline version of the comet assay has been used to determine the effect of tramal administration  (200 and 400mg/Kg) on peroxide-initiated free radical-mediated DNA damage in rat liver cells. Indeed, levels of strand breaks in rat liver cell exposed to tramal400mg/Kg were significantly higher than in cells exposed to 200mg/Kg, especially after a long administration period (TR45 days). The intensity of the comet tail relative to the head reflects the number of DNA breaks. The rates of tailed cells detected by the comet assay increased significantly when the rats were exposed to 200 and 400mg/kg of tramal compared with control (however, the tail length did not differ significantly between the same groups). The intensity of the comet tail relative to the head reflects the number of DNA breaks.   Conclusions: Our findings pointed out the risk of increased lipid peroxidation, hepatic DNA damage and abnormality in serum protein pattern due to long term use of tramadol, although opioids are reported to be effective in pain management, their toxic effects should be kept in mind.