For native breast cancer cell growth to be mimicked in vitro as spheroids, a well-defined matrix that mimics the tumor microenvironment is required. Finding a biomimetic material for 3D cell culture other than Matrigel has challenged the field. Because hyaluronan is naturally abundant in the tumor microenvironment and can be chemically modified, we synthesized a hyaluronan (HA) hydrogel with independently tunable mechanical and chemical properties for 3D culture of breast cancer cells. By modifying HA with distinct bioorthogonal functional groups, its mechanical properties are controlled by chemical cross-linking via oxime ligation, and its biochemical properties are controlled by grafting bioactive peptides via Diels-Alder chemistry. A series of hydrogels were screened in terms of stiffness and peptide composition for cancer spheroid formation. In the optimal hydrogel formulation, the 3D breast cancer spheroids showed decreased drug diffusion into their core and upregulation of cellular multidrug-resistant efflux pumps similar to what is observed in drug-resistant tumors. Our results highlight the potential of these tunable and well-defined gels in drug screening assays.