Synapses are the basic unit of information transfer between neurons. Their dysfunction is a common trigger of cognitive diseases and disorders. However, high-throughput analysis methods to assess synaptic function and dysfunction are lacking. Calcium imaging in cultured neurons in the absence of Mg2+ and presence of TTX allows visualization of NMDAR-dependent spontaneous synaptic calcium transients, which report pre and postsynaptic function. Here, we introduce a high-throughput automated analys