Self assembled peptide hydrogels have potential use in drug delivery and tissue engineering applications. We present a family of de novo peptide designs that links the intramolecular folding of amphiphilic β-hairpin peptides to their propensity to self assemble, affording hydrogel materials. These peptides adopt a random coil conformation in aqueous pH 7.4 solutions and are freely soluble. However, when a physiological relevant concentration of NaCl (150 mM) is added, the peptides fold into a β-hairpin, and subsequently, self-assemble to form a rigid hydrogel stabilized by non-covalent cross-links. For these peptides, it is possible to control the folding and assembly kinetics to form hydrogels with different rigidities. Changing the peptide sequence influences the rate of folding and self-assembling, which modulates the hydrogel stiffness. Alternatively, the rigidity of the resultant material can be increased by increasing the peptide concentration and thereby, incorporating more fibrils into the network as well as hastening the self-assembling kinetics. As these physiochemical changes affect the porous morphology within the hydrogel system, they also affect the rate of macromolecular diffusion within the peptide fibrillar network. Here, we investigated the influence of the gel network of two peptide sequences of varying concentrations on the mobility of biomacromolecules for delivery. Fluorescence recovery after photo-bleaching (FRAP) was used to measure the diffusion coefficients of FITC-dextran macromolecules in the hydrogel. The importance of the interactions between the probes and the hydrogel matrices are further demonstrated in release studies with the FITC-dextrans. Experiments indicate that macromolecule mobility within and release out of these gels can be predictably modulated by either varying the concentration of peptide used to construct the gel or by varying the charge state of the peptide by altering its sequence. By understanding the effect of material properties on the movement of multiple sized species within hydrogel, better and specific design of self-assembling peptide materials can be achieved, allowing for controlled therapeutic delivery for many biomaterial applications.