THE photostimulation of nerve cells using a caged compound is very useful because it is non-invasive and non-destructive compared with standard electrophysiological techniques. There are no methods, however, for continuously measuring the photo-uncaged ‘free’ compound concentration at high temporal and spatial resolutions which can detect how much uncaged compound has been applied to cells. Here, we used an electrochemical detection method for the real-time measurement of photo-uncaged glutamate. In this way, we were able to determine the amount of uncaged glutamate and investigate neural activities by tracing [Ca]i while simultaneously employing photostimulation and on-line glutamate measurement. The combination of an on-line sensor and laser-photostimulation with [Ca]i measurement could be a powerful tool with which to investigate synaptic connections and activities.