Summary Human neurochemistry undergoes dramatic changes in early childhood, due to the rapid pace of development. However, techniques for assessing neurochemistry non-invasively by magnetic resonance spectroscopy (MRS) in young children are limited. Spectral editing is the gold-standard for detecting MRS signals from key neurometabolites such as the neurotransmitters glutamate (Glu) and GABA, the neuromodulators N-acetyl aspartyl glutamate (NAAG) and aspartate (Asp), the redox compounds glutathione (GSH) and ascorbate (Asc), the anaerobic glycolysis product lactate (Lac), and the membrane lipid precursor phosphorylethanolamine (PE). Measurement of these lower-concentration metabolites is vital for investigating healthy and disrupted neurodevelopment, including inhibitory dysfunction, oxidative stress, myelination and mitochondrial metabolic disruption. To date, spectral editing has not been applied at large in pediatrics – arguably the arena in which they will be leveraged most powerfully for scientific discovery and clinical value. This project will develop acquisition and analysis tools for multi-metabolite MRS in the pediatric brain, focusing on developing motion-robust acquisitions. It will also acquire reference data for the best-practice analysis of pediatric spectra, including the macromolecular background spectrum and metabolite relaxometry. This project will have a key push-pull interaction with the HEALthy Brain & Child Development (HBCD) Study, a 25-site national study of neurodevelopmental risk factors (including four sites in this proposal). HBCD applies the acquisition and analysis methods developed by PI Edden's group (HERCULES and Osprey), and Drs. Edden and Wisnowski are co-chairs of the HBCD MRS working group. Dissemination of methods from this project will directly support HBCD, and this new focus on methods development for pediatric cohorts will allow us to directly address technical issues as they arise in HBCD. Data acquired during HBCD will supplement the data acquired in this grant as we develop a profound new understanding of the neurometabolic trajectory of early childhood.