Intellectual disability (ID) and autism spectrum disorder (ASD) are frequently associated neurodevelopmental disorders (NDDs), which affect more than 1% of the general popula- tion, and consequently represent a major public health issue. These conditions are characterized by compromised brain and cognitive functions and impaired social behaviors, representing a leading cause of handicap in children. NDDs often result from genetic mutations leading to neurodevelopmental alterations characterized by structural neuronal network defects, which is associated with aberrant synaptic communication and plasticity. Translational research is absolutely central to better understand human brain patholo- gies. Indeed, the genetic analysis of patients associated with the integration of preclinical data obtained from engineered cellular and/or animal models bearing the identified human mutations in their genome, alongside a detailed characterization of the impact that these mutations pose to neuronal development, plasticity and brain connectivity is a promising strategy to envisage the development of novel therapies for NDDs. This volume of translational research methods applied to the neurodevelopmental disease research fields presents advanced technical developments and their applications associated with more classical techniques that are ranging from molecular aspects to integrated research approaches and brain imaging in living rodents. Briefly, Chap. 1 exposes the protocol to deliver shRNA in vitro or in vivo using lentiviral particles to knock down specific protein expression; Chap. 2 presents the different methods to purify synaptosomes from rodent and human brain tissues as well as a way to enrich the preparations in pre- and post-synaptic compartments. Chapter 3 depicts the experimental procedures to use recombinant fluorescent probes to visualize endogenous proteins at the mammalian synapse. Chapter 4 reports an advanced method to characterize specific sub-neuronal populations in the developing brain. Chapter 5 details a CRISPR/Cas9 toolkit to assess either gain or loss of gene function in brain organoids. Chapter 6 is devoted to a quantitative histological assessment method to precisely assess the neuroanatomical pheno- types of mouse brain. Chapter 7 highlights how to use microelectrode array technology for phenotyping stem cell–derived neurodevelopmental disease models. Chapter 8 presents the use of humanized mouse models to investigate human neural development in health and disease. Chapter 9 reports a detailed protocol to use in vivo manipulations to correct the behavioral phenotypes in cognitive disorder mouse lines. Chapter 10 presents the protocols to measure dyadic social interaction in rodent models of NDDs. The detailed method of how metabolomics is used to study brain metabolic signatures associated with NDDs is detailed in Chap. 11. Chapter 12 gives established experimental approaches to genetically engineer macaque models of NDDs and investigates how genetic predisposition may cause neural and functional alterations. Since alterations in neuronal network formation and/or regulation are also centrally involved in other neurological conditions, such as stroke and epilepsy, as well as in neurode- generative disorders and psychiatric diseases, including, but not restricted to, Parkinson’s, Huntington’s and Alzheimer’s diseases, schizophrenia, or depression, the whole range of step-by-step procedures described in this volume will be of broad interest to any novice or expert neuroscientist. Finally, we would like to thank all the contributors for their outstanding contributions to this volume of Neuromethods.