Developmentally neurotoxic chemicals are a diverse set of substances that have the potential to interfere with the normal development of the nervous system, which, if perturbed without compensation, may lead to adverse effects on the normal development of nervous system structures and/or functions. The current OECD test guideline (TG) 426 requires assessing the impact of fetal and postnatal maternal exposure on the development of: physical and developmental landmarks, clinical observations, and behavioural and neuropathological endpoints (OECD 2007). Use of the in vivo test guideline has been limited due to extensive time, resource costs and animal used. This limited testing, coupled with an increasing need to assess the hazards of hundreds of pesticides and thousands of industrial chemicals, has resulted in calls for the development and use of in vitro methods. International efforts have led to the development of the Developmental Neurotoxicity In Vitro Battery (DNT IVB). The purpose of the present document is to provide guidance on the interpretation of data developed with the DNT IVB for use in regulatory decisions. The major aims are to describe the assays that comprise the battery in terms of neurodevelopment, provide criteria that allows evaluation of the relevance of the data to developmental neurotoxicity, and to assist in the determination of the degree of certainty in any positive or negative findings to better inform use of DNT in vitro data in regulatory hazard determinations.