The human brain presents an incredible structural complexity that enables the amazing richness of behavior. The organization of this organ spans multiple scales and requires tools that enable the reconstruction of centimeter-size volumetric blocks with cellular resolution. Here, we overview the methodologies that allow 3D human brain imaging using light-sheet fluorescence microscopy (LSFM). Specifically, we describe the SHORT method that enables the obtainment of optically transparent and macromolecule-permeable human brain tissues, in combination with tridimensional reconstruction using an inverted custom-made light sheet fluorescence microscope, providing the reader with all the preparation steps needed. We review the challenges related to the analysis of the big data generated by the acquisitions, providing guidelines and indications of solutions. Finally, we present an application on a human Broca’s area as an example of the possible use of tissue clearing and LSFM for cellular mapping in 3D.