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Summary: Early-life nutrition fundamentally influences newborn development and health. Here, we present a protocol for nutritional intervention in neonatal rats using the “pup-in-a-cup” artificial rearing system. We describe steps for rat milk substitute preparation, cheek cannulation and maintenance, and nutritional manipulation during the suckling period. This protocol enables investigation into the role of nutritional factors in newborns by artificially rearing rats away from the mother with experimental diets starting at postnatal day 4 for up to 18 days.For complete details on the use and execution of this protocol, please refer to Wang et al.,1 Choudhary et al.,2 and Mu et al.3,4 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.