Astrocytes, the multi-functional glial cells with the most abundant population in the brain, integrate information across their territories to regulate neuronal synaptic and cerebrovascular activities. Astrocytic calcium (Ca 2+) signaling is the major readout of cellular functional state of astrocytes. The conventional two-photon in vivo imaging usually focuses on a single horizontal focal plane to capture the astrocytic Ca 2+ signals, which leaves >80% spatial information undetected. To fully probe the Ca 2+ activity across the whole astrocytic territory, we developed a pipeline for imaging and visualizing volumetric astrocytic Ca 2+ time-lapse images. With the pipeline, we discovered a new signal distribution pattern from three-dimensional (3D) astrocytic Ca 2+ imaging data of mice under isoflurane anesthetic states. The tools developed in this study enable a better understanding of the spatiotemporal patterns of astrocytic activity in 3D space.