Short peptide self-assembled hydrogels are a promising class of soft nanomaterials for drug delivery and regenerative medicine. Here we describe the gelation of tripeptides, consisting of d and l hydrophobic amino acids, in buffer, following a pH switch to 7.4 (i.e., physiological pH). Interestingly, tripeptide analogues consisting of l-only amino acids fail to form a gel under the same conditions. The nanostructure of these self-assembling peptides is investigated by a number of techniques (atomic force microscopy, transmission electron microscopy, and confocal light microscopy) to unveil their architectures. In addition, these self-assembled soft nanomaterials can be potentially used as vehicles to deliver bioactive molecules. For instance, we describe the incorporation into the gel of rhodamine dye as a model compound in a two-step procedure: firstly, the dye is dissolved in the gel precursor solution; secondly, dilution to a final pH of 7.4 triggers self-assembly and gelation of the system. We analyse the effect of dye incorporation within either precursor solution on the secondary structure, nanoarchitecture, and rheological properties of the resulting peptide materials. We also present data concerning dye release kinetics. Dye release is achieved within 48 h, and no burst release is observed. We anticipate that these systems will find biological applications for the delivery of bioactive compounds.