Abstract"Oxidative DNA damage can be induced by reactive oxygen species and free radicals. Reactive Oxygen Species which is induced by oxidative damage plays a key role in DNA damage". The aim of our study is to identify the protective effect of ascorbic acid (AA, vitamin C) on hydrogen peroxide which induced oxidative damage in DNA by using the Comet assay. Lymphocytes pretreated with or without antioxidants, incubated at 37C for 30 minutes, then H2O2 (100µM) was added & incubated again at 37C for 1 hour (60 minutes). Viability of cells was detected by trypan blue stain exclusion method. The decrease in viability brought about by H2O2 when the cells incubated for 60 minutes and the viability was present to be 39±3% from 80±4% and it was highly developed by the found of AA at 100 μM which appeared 72 ± 1%. These findings indicate that the activity of ascorbic acid as an antioxidant is evidenced by its ability to suppress the oxidative effect against H2O2 and protect the lymphocytes. Estimation of comet tail moment and tail length in human lymphocyte treated with 100µM of hydrogen peroxide as positive control showed that 13± 4.5% of the cells showed no DNA damage while the DNA damage from low to very high damage were 19± 3.5%, 12.2± 2.3%, 14± 3.2% and 37± 3.0%, respectively. In contrast, treatment of the cells with 100 µM H2O2in combination with 10, 25,75