Peptide-based hydrogels are promising biocompatible materials for woundhealing, drug delivery, and tissue engineering applications. The physical propertiesof these nanostructured materials depend strongly on the morphologyof the gel network. However, the self-assembly mechanism of the peptidesthat leads to a distinct network morphology is still a subject of ongoingdebate, since complete assembly pathways have not yet been resolved. Tounravel the dynamics of the hierarchical self-assembly process of the modelβ-sheet forming peptide KFE8 (Ac-FKFEFKFE-NH2), high-speed atomic forcemicroscopy (HS-AFM) in liquid is used. It is demonstrated that a fast-growingnetwork, based on small fibrillar aggregates, is formed at a solid–liquidinterface, while in bulk solution, a distinct, more prolonged nanotube networkemerges from intermediate helical ribbons. Moreover, the transformationbetween these morphologies has been visualized. It is expected that this newin situ and in real-time methodology will set the path for the in-depth unravellingof the dynamics of other peptide-based self-assembled soft materials,as well as gaining advanced insights into the formation of fibers involved inprotein misfolding diseases.