Two-photon microscopy enables imaging of calcium signaling at cellular or subcellular resolution up to hundreds of microns deep in the living brain. Changes in the brightness of fluorescent calcium indicators provide a readout of calcium levels over time, affording information about neuronal activity and/or calcium-dependent subcellular signaling. Here, we describe a protocol for repeated two-photon imaging of calcium signals in mice expressing a genetically encoded calcium indicator that have been implanted with a chronic cranial window.