DNA is the target of many reactive molecules and it is prone to be damaged easily. DNA damage may be resulted either from spontaneously during DNA metabolism or effects of some environmental factors. There are repair mechanisms for DNA damage in cell nucleus since the prevention of DNA structure is extremely important for conducting the genetic information. When a heavy damage occurs or repair mechanisms are defective, DNA damage results in inhibition of replication, transcription or protein synthesis in short term, however in long term, this damage may lead to mutation and chromosomal anomalies. DNA damage which is a contributing factor in the pathogenesis of certain diseases such as cancer, diabetes and atherosclerosis, is being evaluated as the biological marker in assessment of chemotherapy and radiotherapy, determining the radiation and xenobiotic mediated genotoxicity as well as follow up of chronical degenerative diseases. Therefore, techniques that enable the sensitive measurement of DNA damage became important recently. Comet assay is a fast, non invasive, sensitive fluorescent technique, which is being used for detecting DNA damage on single cell level and also determining the amount of damage. After its introduction in 1988 as "Alcaline Comet Assay", it has been developed with many modifications and became a workable technique for detecting a variety of DNA damages. As comet assay was being utilized in aging, molecular epidemiology, clinical and genetic toxicology, recently it is placed also in research of apoptosis and oxidative stress-antioxidants. Current review aims to give information about comet assay and its applications as well as help to propagate its use.