BACKGROUND: Exposure to environmental chemicals that interfere with normal estrogen function can lead to adverse health effects, including cancer. High-throughput screening (HTS) approaches facilitate the efficient identification and characterization of such substances. OBJECTIVES: We recently described the development of the E-Morph Assay, which measures changes at adherens junctions as a clinically-relevant phenotypic readout for estrogen receptor (ER) alpha signaling activity. Here, we describe its further development and application for automated robotic HTS. METHODS: Using the advanced E-Morph Screening Assay, we screened a substance library comprising 430 toxicologically-relevant industrial chemicals, biocides, and plant protection products to identify novel substances with estrogenic activities. Based on the primary screening data and the publicly available ToxCast dataset, we performed an insilico similarity search to identify further substances with potential estrogenic activity for follow-up hit expansion screening, and built seven insilico ER models using the conformal prediction (CP) framework to evaluate the HTS results. RESULTS: The primary and hit confirmation screens identified 27 'known' estrogenic substances with potencies correlating very well with the published ToxCast ER Agonist Score (r=+0.95). We additionally detected potential 'novel' estrogenic activities for 10 primary hit substances and for another nine out of 20 structurally similar substances from insilico predictions and follow-up hit expansion screening. The concordance of the E-Morph Screening Assay with the ToxCast ER reference data and the generated CP ER models was 71% and 73%, respectively, with a high predictivity for ER active substances of up to 87%, which is particularly important for regulatory purposes. DISCUSSION: These data provide a proof-of-concept for the combination of in vitro HTS approaches with insilico methods (similarity search, CP models) for efficient analysis of large substance libraries in order to prioritize substances with potential estrogenic activity for subsequent testing against higher tier human endpoints.