The developmental and reproductive effects of endocrine disruption on humans and wildlife rank among the most threatening of all environmental health concerns. Particularly vulnerable to chemical assaults are the hypothalamic-pituitary-gonadal (HPG) axis and the hypothalamic-pituitary-thyroid (HPT) axis of the endocrine system. While the effects of endocrine disrupting chemicals (EDCs) on the HPG axis have been the subject of intense research efforts, with comprehensive elucidation, a lot remains to be clarified on the effects of EDCs on thyroid functions. For instance, there are no clear-cut biomarkers of exposure and effects of thyroid disrupting chemicals (TDCs) in intact organisms. Consequently, a number of in vitro assays have been developed, and are particularly useful for the identification and mechanistic characterization of potential TDCs considering the increasing number of EDCs that are being released into the environment. However, with the in vitro assays, studies suggest that a plausible major mode of action of TDCs, thyroid hormone receptor (THR) agonist activity, is not environmentally relevant. Here, we reviewed in vitro detection of TDCs activities in wastewater and surface waters. Data strongly suggest that cell lines may be responsible for the less frequent detection of THR agonist activity in wastewater and surface water. It was concluded that the development of reporter gene assays with thyroid hormone function related cell lines, is required.