Fast neurotransmitter release plays an important role in neuron communications. The membrane fusion process mediated by SNAREs can be significantly influenced by proteins related to neurodegeneration. In order to understand the molecular mechanism, a series of single molecule biophysical assays have been developed for observing content mixing rather than (or in addition to) lipid mixing at the single-vesicle level. Based on proteoliposomes reconstituted with v- or t-SNARE proteins, people have developed single vesicle-vesicle fusion assays that monitor lipid or content mixing and both lipid and content of individual vesicle-vesicle pairs. By simultaneously observing lipid and content mixing indicators of single vesicle pairs, these assays provide a foundation to investigate the influence of other regulation factors on SNARE-mediated membrane fusion. Furthermore, these single vesicle-vesicle fusion assays have been applied for systematically characterizing proteins related to neurodegeneration (CSPα, α-Syn, and Aβ) during different stages (docking, hemifusion, and full fusion) of SNARE-mediated membrane fusion.