Most, if not all, cellular processes, exemplified by neuroexocytosis, rely on the precise interplay of specific molecules in both space and time. Super-resolution microscopy allows visualization of single-molecule trajectories far below the diffraction limit of visible light, which opens up possibilities for precise spatiotemporal analysis of these cellular processes. While a range of tools have been developed for assessing the spatial distribution of molecules in fixed cell data, the increasing sophistication of single particle tracking (spt) in live cells requires new approaches for spatiotemporal analysis, which can shed light on the nanoscale dynamics of molecular interaction and clustering. In this chapter, we will discuss a recently developed suite of novel tools—NAnoscale SpatioTemporal Indexing Clustering (NASTIC), which uses the overlap of molecular trajectory bounding boxes to establish interaction in space and time, and will describe a workflow to allow the user to use NASTIC to derive spatiotemporal metrics from their own super-resolved live cell trajectory data.