Fixed tissue analyses of synaptic density and connectivity rely on analyses of image stacks containing fluorescence signals. This chapter details image analysis routines that can be implemented to determine connectivity between labeled cell types and procedures for quantification of synaptic density across a tissue volume or specifically within a labeled cell of interest. The chapter details open-source software and practices that can be implemented for these analyses as well as considerations for validation of detected signals, estimation of background noise in images, and determination of non-specific detections.