Activation of cannabinoid receptor 1 (CB1) inhibits synaptic transmission in hippocampal neurons. The goal of this study was to evaluate the ability of benchmark and emerging synthetic cannabinoids to suppress neuronal activity in vitro using two complementary techniques, Ca2+ spiking and multi-electrode arrays (MEAs). Neuron culture and fluorescence imaging conditions were extensively optimized to provide maximum sensitivity for detection of suppression of neural activity by cannabinoids. The neuronal Ca2+ spiking frequency was significantly suppressed within 10 min by the prototypic aminoalkylindole cannabinoid, WIN 55,212–2 (10 μM). Suppression by WIN 55,212–2 was not improved by pharmacological intervention with signaling pathways known to interfere with CB1 signaling. The naphthoylindole CB1 agonist, JWH-018 suppressed Ca2+ spiking at a lower concentration (2.5 μM), and the CB1 antagonist rimonabant (5 μM), reversed this suppression. In the MEA assay, the ability of synthetic CB1 agonists to suppress spontaneous electrical activity of hippocampal neurons was evaluated over 80 min sessions. All benchmark (WIN 55,212–2, HU-210, CP 55,940 and JWH-018) and emerging synthetic cannabinoids (XLR-11, JWH-250, 5F-PB-22, AB-PINACA and MAM-2201) suppressed neural activity at a concentration of 10 μM; furthermore, several of these compounds also significantly suppressed activity at 1 μM concentrations. Rimonabant partially reversed spiking suppression of 5F-PB-22 and, to a lesser extent, of MAM-2201, supporting CB1-mediated involvement, although the inactive WIN 55,212–3 also partially suppressed activity. Taken together, synthetic cannabinoid CB1-mediated suppression of neuronal activity was detected using Ca2+ spiking and MEAs.