Grana Padano (GP) cheese is a renowned PDO Italian cheese whose nutritional characteristics and market price are influenced by the ripening stage. In this work, it was demonstrated that the combined use of untargeted 1H NMR profiling and chemometric analysis can be used as a powerful tool to quantitatively characterize GP ripening and production, focusing on both aqueous and lipid fractions. An initial exploratory analysis revealed substantial variations in the aqueous fraction attributable to aging time, year and season of production. Multivariate analysis was adopted to show these differences, mainly attributable to amino acids. In contrast, the lipid fraction analysis highlighted the impact of production season on fatty acid unsaturation, influenced by feed variations. As regards the production process, this study focuses on the variations induced by bactofugation. In this respect, the aqueous fraction was found to be extensively influenced by this centrifugation step, affecting compounds crucial to organoleptic characteristics. •Lipophilic and hydrophilic parts of the cheese metabolome reflect different aspects of cheese ripening•Scientific assessment of the effects on cheese composition made by bactofugation step•Development of a fast and simple sample preparation for both fractions, aqueous and lipid