A multiple cell tracking method dedicated to the analysis of memory formation in vivo Formation and consolidation of new memories is one of the fundamental characteristics of the brain, responsible for learning and high cognitive behavior. While important, the process isn’t fully understood to the present day and is the subject of various studies, spanning from the activity analysis of individual synapses to the reconstruction of brain connectivity maps. In this work, we propose a bold approach, on which we aim to measure in vivo the activity of every single neuron from the whole Mushroom body (MB) of the Drosophila melanogaster, in a fully automated procedure. After a 3D image acquisition over time of the MB by means of confocal microscopy, an automated detection and tracking of the neurons is performed. The whole process takes place while the fly is awake and subjected to different odor stimulations, so that it is possible to associate the activity patterns at the single cell level to the stimulus that is being received. By comparing the response patterns from flies that were trained and flies that were not trained to associate an odor with an electric shock we identified changes in neuronal activity, providing information on how memory is formed. Beyond the methodological innovation that brought the possibility to track the activity of a large set of single neurons, this work contributed to the current understanding of long term memory formation.