We report here the identification of substrates of the depalmitoylating enzyme PPT1 by quantitative mass spectrometry. We utilized a stringent two-step Acyl Resin-Assisted Capture (Acyl RAC) screen in which increased palmitoylation in vivo in PPT1 knockout is the first selection criteria for substrates. We validated hits by direct depalmitoylation with PPT1 to identify bona fide substrates. Most proteins identified are previously reported to be palmitoylated and the few known PPT1 substrates were validated. Significantly, the screen identified >100 proteins not previously known to be depalmitoylated by PPT1, including a wide range of endocytic, signaling, synaptic adhesion, mitochondrial, and lysosomal proteins. These proteins collectively explain the many facets of CLN1 disease caused by the loss of PPT1 function. We determined that an Ig domain in synaptic adhesion molecules may be a PPT1 recognition site for depalmitoylation. Finally, we provide evidence that PPT1 participates in neuronal activity-dependent palmitoylation-depalmitoylation cycles at the synapse.