The U.S. Environmental Protection Agency Endocrine Disruptor Screening Program and the Organization for Economic Co-operation and Development (OECD) have used the human adrenocarcinoma (H295R) cell-based assay to predict chemical perturbation of androgen and estrogen production. Recently, a high-throughput H295R (HT-H295R) assay was developed as part of the ToxCast program that includes measurement of 11 hormones, including progestagens, corticosteroids, androgens, and estrogens. To date, 2012 chemicals have been screened at 1 concentration; of these, 656 chemicals have been screened in concentration-response. The objectives of this work were to: (1) develop an integrated analysis of chemical-mediated effects on steroidogenesis in the HT-H295R assay and (2) evaluate whether the HT-H295R assay predicts estrogen and androgen production specifically via comparison with the OECD-validated H295R assay. To support application of HT-H295R assay data to weight-of-evidence and prioritization tasks, a single numeric value based on Mahalanobis distances was computed for 654 chemicals to indicate the magnitude of effects on the synthesis of 11 hormones. The maximum mean Mahalanobis distance (maxmMd) values were high for strong modulators (prochloraz, mifepristone) and lower for moderate modulators (atrazine, molinate). Twenty-five of 28 reference chemicals used for OECD validation were screened in the HT-H295R assay, and produced qualitatively similar results, with accuracies of 0.90/0.75 and 0.81/0.91 for increased/decreased testosterone and estradiol production, respectively. The HT-H295R assay provides robust information regarding estrogen and androgen production, as well as additional hormones. The maxmMd from this integrated analysis may provide a data-driven approach to prioritizing lists of chemicals for putative effects on steroidogenesis.