Synapses are crucial to brain function and frequent disease targets, but current analysis methods cannot report on individual synaptic components in situ or present barriers to widespread adoption. SEQUIN was developed to address this challenge. SEQUIN utilizes a widely available super-resolution platform in tandem with image processing and analysis to quantify synaptic loci over large regions of brain and characterize their molecular and nanostructural properties at the individual and population level. This protocol describes quantification of synaptic loci using SEQUIN. For additional details on the use and execution of this protocol, please refer to Sauerbeck et al. (2020).