Two-photon imaging is a powerful method to record the activity of neuronal ensembles in the intact animal brain. Here, we describe a protocol to run a recent analytical approach, CITE-On, specifically designed for online morphological cell detection, segmentation, and signal extraction in two-photon functional imaging recordings. A background introduction and the description of the main challenges CITE-On was conceived to overcome are presented, together with a detailed experimental procedure to install CITE-On and run it on two-photon calcium imaging time-series (t-series). By performing fast image segmentation, CITE-On will facilitate the establishment of efficient close-loop experimental approaches, including all-optical two-photon imaging and perturbation experiments.