Significance Although synaptic vesicle fusion plays a crucial role in neurotransmission, studies of its molecular mechanisms have relied heavily on the use of artificial liposomes reconstituted with recombinantly expressed proteins. In this study, we establish an in vitro assay using engineered yeast vacuoles bearing neuronal SNAREs for measuring neuronal SNARE-mediated membrane fusion and show that this assay provides a simple and independent means of investigating synaptic vesicle fusion mechanisms.