The steroidogenesis assays can be performed based on interference of chemicals with biosysnthesis of steroid hormones. Although the assays can use animals, ex vivo model, primary cells or cell lines, recently H295R cell based steroidogenesis assay has been validated by OECD and US EPA. In this presentation, inter-validation results and processes will be introduced. Since H295R cells that are derived from human female adrenocortical carcinoma, produce variable steroid hormones such as progestin, testosterone and estradiol and express most of the important steroidogenic enzymes like CYP11A, CYP17 and CYP21, H295R cell based steroidogenesis assay was recommended by US EPA(2005) and standardized by Dr. Heckers M(2006). The pre-validation of this assay was conducted (2006-2007) and validation study was finished by participation of 7 countries, 7 laboratories including Korea FDA. Korea FDA performed the mission of transferability in this validation study because it has never experienced the assay. Coded blind test was carried out 12 core chemicals including strong inducer, Forskolin and strong inhibitor, prochloraz in the validation study. According to the results of pre- and validation study, it could be demonstrated that the H295R steroidogenesis assay represents a powerful screening tool to especially evaluate the effects of chemicals on the biosynthesis of testosterone and estradiol. The assay is also highly convenient and cost effective and may reduce the use of experimental animals. Recently US EPA non Animal group has reviewed the report of validation results and submitted draft Test Guideline for H295R cell based steroidogenesis assay to OECD(2009).