Ionic Zn2+ has increasingly been recognized as an important neurotransmitter and signaling ion in glutamatergic neuron pathways. Intracellular Zn2+ transiently increases as a result of neuronal excitation, and this Zn2+ signal is essential for neuron plasticity, but the source and regulation of the signal is still unclear. In this study we rigorously quantified Zn2+, Ca2+ and pH dynamics in dissociated mouse hippocampal neurons stimulated with bath application of high KCl or glutamate. While both stimulation methods yielded Zn2+ signals, Ca2+ influx, and acidification, glutamate stimulation induced more sustained high intracellular Ca2+ and a larger increase in intracellular Zn2+. However, the stimulation-induced pH change was similar between conditions, indicating that a different cellular change is responsible for the stimulation-dependent difference in Zn2+ signal. This work provides the first robust quantification of Zn2+ dynamics in neurons using different methods of stimulation.