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A major challenge in the use of HepG2 cell culture models for drug toxicity screening is their lack of maturity in 2D culture. 3D culture in Matrigel promotes the formation of spheroids that express liver-relevant markers, yet they still lack various primary hepatocyte functions. Therefore, alternative matrices where chemical composition and materials properties are controlled to steer maturation of HepG2 spheroids remain desired. Herein, a modular approach is taken based on a fully synthetic and minimalistic supramolecular matrix based on squaramide synthons outfitted with a cell-adhesive peptide, RGD for 3D HepG2 spheroid culture. Co-assemblies of RGD-functionalized squaramide-based and native monomers resulted in soft and self-recovering supramolecular hydrogels with a tunable RGD concentration. HepG2 spheroids are self-assembled and grown (≈150 µm) within the supramolecular hydrogels with high cell viability and differentiation over 21 days of culture. Importantly, significantly higher mRNA and protein expression levels of phase I and II metabolic enzymes, drug transporters, and liver markers are found for the squaramide hydrogels in comparison to Matrigel. Overall, the fully synthetic squaramide hydrogels are proven to be synthetically accessible and effective for HepG2 differentiation showcasing the potential of this supramolecular matrix to rival and replace naturally-derived materials classically used in high-throughput toxicity screening.