The invention of protein-based fluorescent biosensors has paved the way to target specific cells with these probes and visualize intracellular processes not only in isolated cells or tissue cultures but also in transgenic animals. In particular, DNA-encoded fluorescence proteins sensitive to Ca(2+) ions are often used to monitor changes in intracellular Ca(2+) concentrations. This is of particular relevance in neuroscience since the dynamics of intracellular Ca(2+) concentrations represents a faithful correlate for neuronal activity, and optical Ca(2+) imaging is commonly used to monitor spatiotemporal activity across populations of neurons. In this respect Drosophila provides a favorable model organism due to the sophisticated genetic tools that facilitate the targeted expression of fluorescent Ca(2+) sensor proteins. Here we describe how optical Ca(2+) imaging of neuronal activity in the Drosophila brain can be carried out in vivo using two-photon microscopy. We exemplify this technique by describing how to monitor odor-evoked Ca(2+) dynamics in the primary olfactory center of the Drosophila brain.