Helical intermediates appear crucial in amyloid formations of Aβ peptide, α-synuclein, and islet amyloid polypeptide (IAPP, amylin), which are implicated in Alzheimer’s disease, Parkinson’s disease, and Type 2 diabetes, respectively. Many research papers have reported that the intermediate species are more toxic than mature amyloid fibrils. This has narrowed the focus to understand the structural and mechanistic role of intermediates in amyloid self-assembly and their cellular toxicity. Here, we have used an amphibian peptide, uperin 3.5, that is both antimicrobial and amyloidogenic to investigate the structural changes that lead to amyloid formation. The uperin peptides acquire a variety of secondary structure changes during self-assembly into amyloid-rich fibrils under physiological conditions. 1,2 Microsecond time-scale molecular dynamics (MD) simulations reveal the role of the helical intermediates leading to β-sheet-rich aggregates. We show that unstable, partial α-helical peptides, play a key role in beta-sheet-rich aggregation processes. Uperin peptides, with partial helical structures, aggregate to form small clusters, called helical intermediates, via hydrophobic interactions in the initial stages of aggregation. This leads to an increase in the local peptide concentration, which triggers the β-sheet transition in these aggregates. Furthermore, electrostatic interactions between aspartic acid (4th residue) and arginine (7th residue) drive the initial 310-helix formations that further transform into α-helices and, thereby the helical intermediates. An energy landscape of various conformers shows that the helical intermediate is on-pathway of β-sheet aggregation. Hydrogen bond and contact map analysis reveal that the helical intermediates facilitate the inter-peptide interactions by means of increased local peptide concentration. The helical intermediates can be a good candidate in the development of therapeutics against amyloid-associated diseases.