Long-term depression (LTD), a key form of synaptic plasticity, is typically induced through regulated Ca(2+) entry via NMDA receptors and achieved by prolonged (up to hundreds of seconds) low-frequency presynaptic stimulation or bath application of NMDA receptor agonists. Electrophysiological approach to LTD induction requires specialized equipment, while bath applications limit productivity, as only one neuron per sample may be recorded. Here, we present a simple and effective protocol for pharmacological modeling of LTD in primary cultured neurons. This approach relies on highly localized iontophoretic application of NMDA, which induces LTD in individual cells, enhancing experimental throughput. We have analyzed spatio-temporal patterns of iontophoretic drug delivery and demonstrated how this technique may be combined with electrophysiological and live-cell imaging approaches to investigate LTD-related changes in synaptic strength and Ca(2+)-dependent signaling of neuronal Ca(2+) sensor proteins. Key features • Easy, fast, and reliable induction of LTD in primary cultured neurons using iontophoretic NMDA application. • Suitable for the application of any ionic water-soluble compound and compatible with simultaneous multicolor fluorescence imaging and electrophysiological recording. • This protocol enables pharmacological targeting of individual neurons, substantially increasing experimental throughput.