Protein-mediated fusion between phospholipid bilayers is a fundamental and necessary mechanism for many cellular processes. The short-lived nature of the intermediate states visited during fusion makes it challenging to capture precise kinetic information using classical, ensemble-averaging biophysical techniques. Recently, a number of single-particle fluorescence microscopy-based assays that allow researchers to obtain highly quantitative data about the fusion process by observing individual fusion events in real time have been developed. These assays depend upon changes in the acquired fluorescence signal to provide a direct readout for transitions between the various fusion intermediates. The resulting data yield meaningful and detailed kinetic information about the transitory states en route to productive membrane fusion. In this review, we highlight recent in vitro and in vivo studies of membrane fusion at the single-particle level in the contexts of viral membrane fusion and SNARE-mediated synaptic vesicle fusion. These studies afford insight into mechanisms of coordination between fusion-mediating proteins as well as coordination of the overall fusion process with other cellular processes. The development of single-particle approaches to investigate membrane fusion and their successful application to a number of model systems have resulted in a new experimental paradigm and open up considerable opportunities to extend these methods to other biological processes that involve membrane fusion.