Understanding the assembly of small molecules in aqueous media is crucial for the development of adaptive biomaterials. The mechanical properties of supramolecular networks, including stiffness and stress relaxation, play a key role in cellular spreading and can be tuned via formulation strategies or monomer design. Here, we demonstrate the modulation of supramolecular polymerization and cellular response of ureidopyrimidinone (UPy) monomers in water by tailoring the length of the alkyl spacer within the monomer structure. A library of four UPy derivatives with varying hydrophilic-hydrophobic balances was synthesized by using an optimized synthetic approach. The assembly behavior and dynamics of the supramolecular polymers were investigated both in solution and gel states using a wide range of techniques. The results revealed that the alkyl spacer length significantly affects the supramolecular polymer dynamics, kinetics, and stability. Monomers with 6 and 8 methylene units formed dynamic elongated structures, while those with 10 and 12 units yielded robust and stable bundled fibers. In the gel state, a physical cross-linker was required for gel formation. The gels formed by the monomers featuring 8 and 10 methylene units exhibited optimal mechanical properties, promoting the spreading of human normal dermal fibroblasts in both 2D and 3D cultures. These findings highlight the impact of the monomer architecture on the properties of UPy supramolecular systems, paving the way for the rational design of biomaterials with tunable properties.