3D rat brain aggregate model is an excellent tool for mechanistic studies including OMICS analysis. However, their metabolic profile have not been yet fully investigated. The aim was to investigate by 1H-HR-MAS NMR the metabolic fingerprint of 3D brain cell cultures at different maturation stages to establish a developmental profile of metabolic changes. Chemometric analysis revealed a clear separation of samples from the different maturation days. Metabolite concentration evolutions could be followed and revealed strong and various metabolic alterations. The strong metabolite evolution emphasizes the brain modelling complexity during maturation, possibly reflecting physiological processes of brain tissue development.