The LaVision BioTec UltraMicroscope II is a light-sheet microscope ideally suited for imaging of large, fixed, cleared, fluorescent samples, with cellular resolution. This guide describes the basic principles of this system’s operation, the main configurations available, how to minimize risks when using it, tips for sample preparation and mounting and an extensive discussion of how to optimize imaging parameters. It also provides advice for how to handle data transfers from the microscope, as well as how to configure a workstation for data visualization and basic analysis. Finally, it provides standard operating procedures that can be used with this system in other labs or core facilities. This guide aims to provide a baseline of practical information and recommended workflows for new users of this system.