Endocrine-disrupting chemicals (EDCs) interfere with physiological function by mimicking or blocking hormones; these chemicals enter the body through food and its associated pathways and disrupt the endocrine system. Thyroid hormones (THs) control various physiological functions. Despite the increasing threat of endocrine-disrupting chemicals (EDCs) affecting THs, the development of effective testing method detecting and evaluating thyroidal EDCs is insufficient. Therefore, it is necessary to develop an in vitro TH agonist transactivation (TA) assay detecting thyroidal EDCs. In this study, human cell -based transactivation assay that overcame complexity of thyroid transactivation processes was developed. Furthermore, the developed TH agonist TA assay was proved the test method that can provide information regarding the toxicity of specific chemicals absenting thyroid hormone endocrine disrupting activity information by testing the chemical substances recommended by Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and Organisation for Economic Co-operation and Development (OECD). TH agonist TA assay was developed by using the adenocarcinoma human alveolar basal epithelial cell line (A549) and the human cervix adenocarcinoma cell line (HeLa). To solve the problem of measurement over time, which is a disadvantage of the existing assay, a secreted luciferase system was introduced, and GFP was introduced to secure convenience and accuracy in cell line selection. LHCX-hTRE-secNluc-EGFP vector was constructed producing retrovirus harboring hTRE-secNluc-EGFP dual reporter systems. All the prepared vectors were used for cell line test method development after confirming successful cloning operation. This retrovirus harboring hTRE-secNluc-EGFP dual reporter system was usefully used to overcome the problems of existing assays by stably expressing TR, secreted luciferase, and EGFP in response to T3 or EDCs. In addition, optimal conditions of the assay were established by optimization processes to establish in vitro assay using stable cell line. Depending on the established assay conditions, TH agonistic activity of 17 chemicals was evaluated. The 17 chemicals were selected out of Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) recommended chemicals (78 chemicals) and OECD Guidance Documents No. 207 (OECD GD 207) to validate the method. As with previously reported studies, five chemicals showed that the TH agonistic activity. TH agonist TA assay using HeLa cell line developed in this study was confirmed in terms of accuracy and reproducibility with replication tests. In addition, the results suggested that the TH agonist TA assay developed in this study using a human cell line can be used as a reliable screen method screening chemicals with potential TR agonistic activities.