Developmental neurotoxic chemicals are a diverse set of substances that have the potential to interfere with the normal development of the human nervous system. The potential for exposure to environmental chemicals to have adverse impacts on neurodevelopment led to the development of in vivo testing batteries starting in the 1980s. The current in-vivo testing paradigm for developmental neurotoxicity (DNT) is based on OECD test guideline (TG) 426 (or OECD TG 443 with the inclusion of a DNT arm) for in vivo studies where motor and sensory function, learning and memory (only in OECD TG 426), and neuropathology (including morphometric assessment) following maternal exposure are screened. Understanding the methods used in these assays as well as the biological significance and interpretation of the measured endpoints has been recognised as particularly challenging. A NAFTA Technical Working Group therefore developed DNT guidance to help with conducting such studies and interpreting their results. The training course organised by EFSA was intended to provide insights on the principles reported in the guidance document, and give participants the opportunity to listen to the scientists that participated in the guidance development and ask them questions.