Currently, humans and other living things can have many genotoxic damages due to reasons such as chemicals, drugs, unfavorable environmental effects. Breaks in the DNA structure due to genotoxic damage can cause mutations, changes in chromosome structure and cancer. Comet assay, also known as a single cell gel electrophoresis (SCGE), is a sensitive, rapid and cheap method used to measure DNA damage and repair at the individual cell level. With this method, cells are called this way because of they appear as comets under a microscope. DNA damage and repair can be detected in each cell type that can be obtained as single cell suspension, with comet assay. In addition to the direct determination of DNA damage in a single cell, it is possible to determine whether all cells in a population suffer from the same amount of damage. Furthermore, this assay may help to predict the heterogeneous response of cells during any treatment, and the tumor response in radiotherapy and chemotherapy treatment protocols. Especially in human biomonitoring studies, it is also used in the investigation of DNA damage in people who exposed to various occupational or accident-related environmental or workplace-related agents. In this review, a general overview of the comet assay and its currently applications to determine the genotoxicity of environmental factors and chemicals are discussed.