This chapter describes how to apply two-photon neuroimaging to study the insect olfactory system in vivo. It provides a complete protocol for insect brain functional imaging, with some additional remarks on the acquisition of morphological information from the living brain. We discuss the most important choices to make when buying or building a two-photon laser-scanning microscope. We illustrate different possibilities of animal preparation and brain tissue labeling for in vivo imaging. Finally, we give an overview of the main methods of image data processing and analysis, followed by a short description of pioneering applications of this imaging modality.