Astrocytes are the predominant nonneuronal cell type in the central nervous system. Although they are electrically nonexcitable, they have been found to play an active role in modulation of neuronal function and plasticity through Ca2+ excitability. Thus, Ca2+ signaling in astrocytes serves as a mediator of bidirectional interactions between neurons and astrocytes. Although astrocytic Ca2+ signaling has been extensively studied in cultured cells, the recent development of two-photon laser scanning fluorescent microscopy and astrocyte-specific dye labeling make it possible to study astrocytic Ca2+ signaling in live animals. Here we describe a detailed protocol for in vivo Ca2+ imaging of astrocytes in mice.