MPScope is a software suite to control and analyze data from custom-built multiphoton laser scanning fluorescence microscopes. The acquisition program MPScan acquires, displays and stores movies, linescans, image stacks or arbitrary regions from up to four imaging channels and up to two analog inputs, while plotting the intensity of regions of interest in real-time. Bidirectional linescans allow 256×256 pixel frames to be acquired at up to 10fps with typical galvanometric scanners. A fast stack mode combines movie acquisition with continuous z-focus motion and adjustment of laser intensity for constant image brightness. Fast stacks can be automated by custom programs running in an integrated scripting environment, allowing a 1mm³ cortical volume to be sampled in 1billion voxels in approximately 1h. The analysis program MPView allows viewing of stored frames, projections, automatic detection of cells and plotting of their average intensity across frames, direct frame transfer to Matlab, AVI movie creation and file export to ImageJ. The combination of optimized code, multithreading and COM (Common Object Model) technologies enables MPScope to fully take advantage of custom-built two-photon microscopes and to simplify their realization.