The AML12 mouse hepatic cell line presents a promising alternative to primary hepatocytes for hepatic metabolism studies, given the challenges of primary cell availability, donor variability, and rapid dedifferentiation. This protocol optimizes the culture conditions of AML12 cells to better replicate physiological insulin levels and growth factors. We developed a treatment medium (Tx-media) with reduced insulin (1 ng/mL), transferrin (0.55 ng/mL), selenium (0.0005 ng/mL), and no dexamethasone. AML12 cells cultured in Tx-media exhibited enhanced insulin sensitivity, reduced translation burden, and improved bioenergetics compared to cells in complete or ITS-negative media. This optimized culture method significantly improves the metabolic relevance of AML12 cells, making them a robust and reproducible model for studying hepatic metabolism, insulin signaling, and related metabolic disorders and drug screening, bridging the gap between in vitro and in vivo hepatic research.