The synthesis and supramolecular properties of novel glyoxylamide-based short peptides formed via the ring-opening reaction of N-acetylisatins in solution phase are described. The short peptides self-assembled into gels, which were examined for their mechanical and morphological characteristics using multiple spectroscopic and microscopy techniques. The critical gel concentration and mechanical strength of the self-assembled gels were influenced by the presence of electronegative substituents (such as fluoro, in 5b) or hydrophobic substituents (such as bromo, 5d) respectively in the short peptides. Moreover, in vitro cytotoxicity assays demonstrated that these compounds were non-toxic to mammalian cells.