Nanoimaging methods, atomic force microscopy (AFM) in particular, are widely used to study the interaction of biological molecules with the supported lipid bilayer (SLB), which itself is a traditional model for cellular membranes. Success in these studies is based on the availability of a stable SLB for the required observation period, which can extend several hours. The application of AFM requires that the SLB have a smooth morphology, thus enabling visualization of proteins and other molecules on its surface. Herein, we describe protocols for SLB assembly by using 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS) on a mica support. Our methodology enables us to assemble defect-free POPC and POPS SLBs that remain stable for at least 8 h. The application of such smooth and stable surfaces is illustrated by monitoring of the on-surface aggregation of amyloid proteins with the use of time-lapse AFM.