The cerebellar cortex contains two astrocyte types: the Bergmann glia of the molecular layer and the velate protoplasmic astrocytes of the granule cell layer. In vivo, these cell types generate both subcellular calcium transients and trans-glial calcium waves. It is possible to perform in vivo calcium imaging in cerebellar astrocytes. One method involves injection of a replication-incompetent recombinant adenovirus for gene transfer of a fluorescent calcium indicator protein. A second method uses multicell bolus loading (MCBL) in the molecular layer of the cerebellum with synthetic calcium indicators. This protocol presents a cerebellar craniotomy procedure which can be used to prepare a virus-injected animal for in vivo imaging. It can also be used to prepare an animal for MCBL.