Analysis of the pore formation mechanisms of biological nanopores can provide insight into pore-forming peptide-induced diseases and into the characterization of nanopores employed in sensing methods. Evaluation of pore formation mechanisms is typically performed using microscopy including atomic force microscopy, transmission electron microscopy, as well as electrically via channel current measurements using a patch-clamp amplifier. However, due to the relatively low temporal resolution of the above-mentioned microscopy techniques and the low analysis accuracy of the channel current measurements, new analytical methods are required. Here, we describe a new analytical strategy to measure and analyze both ionic currents associated with biological nanopore insertion and deinsertion into and out of lipid bilayers to determine pore formation mechanisms for several representative proteins. The current changes associated with protein deinsertion are monitored as the lipid membrane leaflets are pulled apart-a unique phenomenon enabled by our gold nanoneedle measurement probe. This deinsertion current analysis (DiCA) is performed using a gold nanoneedle-supported lipid bilayer at which a bilayer membrane is formed by bringing together two lipid monolayers on the surface of the nanoneedle and at the interface of an aqueous solution and a lipid/oil mixture. The lipid bilayer can be pulled apart by removing the nanoneedle from this interface. In this study, we demonstrate the determination of pore formation mechanisms for four different pore-forming proteins and peptides-alpha-hemolysin, streptolysin O, alamethicin, and amyloid beta 1-42 using DiCA. As a result, we successfully discern the pore formation mechanism, either addition or expansion, for each protein/peptide by analyzing the ratio and magnitude of insertion and deinsertion current events.