Synaptic neurotransmission is an adaptive process in which synapses exhibit short- and long-term plasticity, dynamically strengthening or weakening their output in response to stimulation. Central to this fine-tuning is the reorganization of synaptic machinery and architecture, with nanoscale alterations in receptor clustering, vesicle trafficking and cytoskeletal rearrangements taking place over a short time frame. Traditionally, visualizing the dynamic organization of the synapse through confocal imaging has been challenging, considering the synapse’s small size and the resolution limits imposed by light diffraction. The emergence of super-resolution imaging, and especially single-molecule localization microscopy (SMLM), over the last couple of decades, has allowed scientists to achieve nanoscale resolution and start unravelling the molecular underpinnings of synaptic transmission and plasticity in real-time. One such approach is single particle tracking Photo-Activated Localization Microscopy (sptPALM), which takes advantage of total internal reflection fluorescence (TIRF) and sparse, stochastic activation of fluorophores to achieve low spatiotemporal density labelling required to track single synaptic proteins in real time. In this chapter, we provide a conceptual breakdown of sptPALM: its key principles, implementation, and impact on the field of neurotransmission. Further, we detail a systematic protocol for performing sptPALM imaging of synaptic proteins in primary neuronal cultures. This technique allows the experimenter to determine the mobility, cluster patterns, and behavior of the synaptic proteins with nanoscale localization precision.