This study presents the development of human trophoblast stem cell-based organoid models that recapitulate the structure and function of the human placental barrier. The organoids are generated from human trophoblast stem (TS) cells and form spherical structures with a single layer of syncytiotrophoblast (ST) cells that exhibit barrier function. The ST cells are derived from TS cells through a three-step culture protocol involving PreM, W-DM, and S-DM media. The organoids are then used to create a column-type ST barrier model, which allows for the measurement of transepithelial/transendothelial electrical resistance (TEER), secretion of human chorionic gonadotropin (hCG), and drug permeability. The model is validated using model compounds such as antipyrine, caffeine, and glyphosate, and shows similar permeability trends to ex vivo perfusion systems. The model also allows for the evaluation of compound transfer and toxicity, which will facilitate drug development. The study highlights the potential of these organoids as a valuable tool for studying placental development and the transplacental passage of xenobiotics. The models are also useful for evaluating the effects of maternal molecules such as IgG and IgA on the placental barrier. The study demonstrates that the organoids can be used to generate a stable ST barrier model with high ST coverage, which is essential for accurately assessing the transplacental passage of chemicals. The models are also applicable to the study of placental pathology and the development of new drugs. The study provides a new approach for the development of in vitro placental barrier models that can be used to assess the transplacental passage of chemicals and the effects of maternal molecules on the placental barrier. The models are also useful for studying the effects of various pathogens and bacteria on the placental barrier. The study shows that the organoids can be used to generate a stable ST barrier model with high ST coverage, which is essential for accurately assessing the transplacental passage of chemicals. The models are also useful for studying the effects of various pathogens and bacteria on the placental barrier. The study provides a new approach for the development of in vitro placental barrier models that can be used to assess the transplacental passage of chemicals and the effects of maternal molecules on the placental barrier.This study presents the development of human trophoblast stem cell-based organoid models that recapitulate the structure and function of the human placental barrier. The organoids are generated from human trophoblast stem (TS) cells and form spherical structures with a single layer of syncytiotrophoblast (ST) cells that exhibit barrier function. The ST cells are derived from TS cells through a three-step culture protocol involving PreM, W-DM, and S-DM media. The organoids are then used to create a column-type ST barrier model, which allows for the measurement of transepithelial/transendothelial electrical resistance (TEER), secretion of human chorionic gonadotropin (hCG), and drug permeability. The model is validated using model compounds such as antipyrine, caffeine, and glyphosate, and shows similar permeability trends to ex vivo perfusion systems. The model also allows for the evaluation of compound transfer and toxicity, which will facilitate drug development. The study highlights the potential of these organoids as a valuable tool for studying placental development and the transplacental passage of xenobiotics. The models are also useful for evaluating the effects of maternal molecules such as IgG and IgA on the placental barrier. The study demonstrates that the organoids can be used to generate a stable ST barrier model with high ST coverage, which is essential for accurately assessing the transplacental passage of chemicals. The models are also applicable to the study of placental pathology and the development of new drugs. The study provides a new approach for the development of in vitro placental barrier models that can be used to assess the transplacental passage of chemicals and the effects of maternal molecules on the placental barrier. The models are also useful for studying the effects of various pathogens and bacteria on the placental barrier. The study shows that the organoids can be used to generate a stable ST barrier model with high ST coverage, which is essential for accurately assessing the transplacental passage of chemicals. The models are also useful for studying the effects of various pathogens and bacteria on the placental barrier. The study provides a new approach for the development of in vitro placental barrier models that can be used to assess the transplacental passage of chemicals and the effects of maternal molecules on the placental barrier.