The properties of a hydrogel utilized in 3D culture can influence cell phenotype and morphology, yielding striking similarities to cellular processes that occur in vivo . Indeed, research areas including regenerative medicine, tissue engineering, cancer models, and stem cell cultures have readily utilized 3D biomaterials to investigate cell biological questions. However, cells are only one component of this milieu. Macromolecules play roles as bioactive factors and physical structures. Yet, investigations of macromolecular biophysics largely focus on pure molecules in dilute solution. Biophysical processes such as protein aggregation underlie diseases including Alzheimer’s disease, which is hallmarked by accumulated neurotoxic amyloid-β (Aβ) aggregates. Previously, we demonstrated that Aβ cytotoxicity is attenuated when cells are cultured within type I collagen hydrogels vs. on 2D substrates. Here, we investigated whether this phenomenon is conserved when Aβ is confined within hydrogels of varying physiochemical properties, notably mesh size and bioactivity. We investigated Aβ structure and aggregation kinetics in solution and in hydrogels (collagen, agarose, hyaluronic acid and polyethylene glycol) using fluorescence correlation spectroscopy and thioflavin T assays. Our results reveal that all hydrogels tested were associated with Aβ cytotoxicity attenuation. We suggest that confinement itself imparts a profound effect, possibly by stabilizing Aβ structures and shifting the aggregate equilibrium toward larger species. It is likely that the milieu that exist within cells and tissues also influences protein-protein interactions; thus, we suggest that it is critical to evaluate whether protein structure, function, and stability are altered in 3D systems vs. ideal solutions and 2D culture.