To understand how membrane proteins function requires characterizing their structure, assembly, and inter- and intramolecular interactions in physiologically relevant conditions. Conventionally, such multiparametric insight is revealed by applying different biophysical methods. Here we introduce the combination of confocal microscopy, force–distance curve-based (FD-based) atomic force microscopy (AFM), and single-molecule force spectroscopy (SMFS) for the identification of native membranes and the subsequent multiparametric analysis of their membrane proteins. As a well-studied model system, we use native purple membrane from Halobacterium salinarum, whose membrane protein bacteriorhodopsin was His-tagged to bind nitrilotriacetate (NTA) ligands. First, by confocal microscopy we localize the extracellular and cytoplasmic surfaces of purple membrane. Then, we apply AFM to image single bacteriorhodopsins approaching sub-nanometer resolution. Afterwards, the binding of NTA ligands to bacteriorhodopsins is localized and quantified by FD-based AFM. Finally, we apply AFM-based SMFS to characterize the (un)­folding of the membrane protein and to structurally map inter- and intramolecular interactions. The multimethodological approach is generally applicable to characterize biological membranes and membrane proteins at physiologically relevant conditions.