Helical wheel projections of peptides based on the repeating unit Ac-(AAKA)n-NH2 clearly illustrate an amphipathic nature. These alanine-based peptides should form helices if the number of residues exceeds a certain threshold value, which was indeed observed for Ac-(AAKA)4-NH2 by Electronic Circular Dichroism (ECD) spectroscopy at milimolar concentrations in aqueous solution. However, these amphipathic peptides, in particular, Ac-(AAKA)3-NH2 and Ac-(AAKA)4-NH2 were found to form hydrogels with an underlying antiparallel Beta-sheet structure at centimolar concentrations, as probed by FTIR, Vibrational Circular Dichroism (VCD), and visible Raman spectroscopy. Atomic Force Microscopy (AFM) images of the 16-mer clearly indicate the formation of amyloid-like fibrils. This aggregation significantly influences the intensity of the amide I' band profile relative to the amide II', when compared with the respective profiles of the previously reported unaggregated octapeptide (AAKA)2. This finding renders the possibility of using amide II' as a marker band for aggregation of alanine-based peptides.