Bioassays (commonly used shorthand for biological assay or assessment) are analytical methods to determine biological hazards of xenobiotics on living animals or plants (in vivo bioassays), or on living cells or tissues (in vitro bioassays). Chemical substances that are not naturally found in the environment and are often introduced as a result of anthropogenic factors are called as xenobiotics. Bioassays determine biological responses against these chemicals that provide essential data for environmental monitoring and risk assessment. This review emphasizes the applications, advantages, challenges, significances and critical roles of bioassays in ecotoxicology, and the advances in its methods. Particularly, it was focused on the significance, advantages and applications of in vivo bioassays in environmental risk assessment by means of exemplifying with various case studies used different taxa of vertebrates as model organisms. These bioassays assessing the effects of xenobiotics on living organisms within their natural or semi-natural environments are crucial tool in ecotoxicology because of their ecological relevance, ability to detect sub-lethal effects and capacity to conduct holistic assessments. Advances in bioassay methodologies have been enhancing the accuracy and efficiency of ecotoxicological assessments contributing to the protection of environmental health and biodiversity against increasing environmental challenges.