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Synapse formation is a fundamental process that shapes the connectivity and function of the nervous system, but the mechanisms regulating synaptogenesis are incompletely understood. Moreover, the interplay of these mechanisms at distinct synapse types remains to be defined. Using a scalable optical pooled screening platform, we investigated the process of synapse induction to uncover modulators of a prototypical synapse-organizing adhesion molecule, neuroligin-1. Analysis of over two million single-cell phenotypic profiles identified 102 candidate regulators of neuroligin-1 that are linked to cell adhesion, cytoskeletal dynamics, and signaling. Among these, we show that the phosphatase PTEN and the dystrophin-associated glycoprotein DAG1 promote neuroligin's roles in inducing presynaptic assembly, with DAG1 selectively regulating inhibitory synapses. This work establishes a scalable high-content screening approach for cell-cell interactions that enables systematic studies of the molecular interactions guiding synaptogenesis.