Therefore, in this chapter, we have discussed the types of ecosystem, genotoxicity versus cytotoxicity, genotoxicity assay types, the importance of genotoxicity assay, comet assay protocol for the identification of genotoxicity, applications of comet assay as a tool in ecogenotoxicology and its application in translational research. A landmark achievement was reached in 2016 when the Organization for Economic Co-operation and Development adopted a comet assay guideline for in vivo testing of DNA strand breaks in animals. Its use has increased significantly in the past few years. Recently, the simple, sensitive, and rapid technique called single-cell gel electrophoresis or comet assay for detecting DNA damage caused by the pollutants in the terrestrial ecosystem models such as earthworms, plants and mammals were developed. During the last 30 years, the comet assay has become widely used for the measurement of DNA damage and repair in cells and tissues. During the last 30 years, the comet assay has become widely used for the measurement of DNA damage and repair in cells and tissues. The comet assay is a sensitive and rapid method for DNA strand break detection in individual cells. Ames assay and micronucleus assay however, these methods have some pitfalls. The genotoxicity was assessed by multiple methods, viz. Hence, there is an urgent necessity to monitor the genetic toxicity in the terrestrial ecosystem or soil-based organism. Due to industrialization and other human activities, organic pollutants accumulated in the soil these accumulated soil pollutants were interacted with the biotic components’ genome and cause genotoxicity in the genetic material and led to various diseases. The soil is the resident of the biotic components of the terrestrial ecosystem. Environmental Chemistry for a Sustainable World