Classical cases of developmental neurotoxicity (DNT) in humans and advances in risk assessment methods did not prevent the emergence of new chemicals with (suspected) DNT potential. Exposure to these chemicals may be related to the increased worldwide incidence of learning and neurodevelopmental disorders in children. DNT is often investigated in a traditional manner (in vivo using large numbers of experimental animals), whereas development of in vitro methods for DNT reduces animal use and increases insight into cellular and molecular mechanisms of DNT. Several essential neurodevelopmental processes, including proliferation, migration, differentiation, formation of axons and dendrites, synaptogenesis, and apoptosis, are already being evaluated in vitro using biochemical and morphological endpoints. Yet, investigation of chemical-induced effects on the development of functional neuronal networks, including network formation, inter- and intracellular signaling and neuronal network function, is underrepresented in DNT testing. This view therefore focuses on in vitro models and innovative experimental approaches for functional DNT testing, ranging from optical and electrophysiological measurements of intra- and intercellular signaling in neural stem/progenitor cells to measurements of network activity in neuronal networks using multielectrode arrays. The development of functional DNT assays will strongly support the decision-making process for measures to prevent potential chemical-induced adverse effects on neurodevelopment and cognition in humans. We therefore argue that for risk assessment, biochemical and morphological approaches should be complemented with investigations of neuronal (network) functionality.