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Synapse loss correlates with cognitive decline in neurodegenerative diseases like late-onset Alzheimer’s disease. We developed a semi-automated workflow to quantify synapse loss in primary mouse neurons. The protocol includes culturing neurons on coverslips, using short hairpin RNA (shRNA)-expressing lentivirus, maintaining cultures, and labeling excitatory and inhibitory presynaptic markers by immunofluorescence. Image acquisition and analysis using Fiji/ComDet macros enable region of interest selection, neurite length measurement, puncta detection, and quantification of synapse density, size, and intensity following Bin1 knockdown. For complete details on the use and execution of this protocol, please refer to Barata et al.