The mammary gland is a dynamic organ whose parenchyma undergoes major development during puberty and extensive remodeling with each estrous cycle. These processes can be modelled and investigated in vitro via 3D cell culture techniques that employ specialized extracellular matrices and appropriate growth factors. The resulting mammary organoid cultures faithfully represent the mammary gland with respect to cellular heterogeneity, cell-cell contacts, overall architecture as well as response to growth factor stimuli and are amendable to a variety of molecular methods as well as microscopy techniques. Among the imaging techniques, light sheet microscopy (single plane illumination microscopy; SPIM) represents a useful method for longitudinal monitoring of morphological changes and cell behavior during the establishment of mammary gland ductal systems. In contrast to other fluorescence microscopy techniques such as widefield- and confocal-microscopy, SPIM exerts minimal phototoxicity while allowing fast acquisition of different fluorophores within organoids arranged in a 3D matrix under optimized environmental conditions. Here, we provide a detailed protocol for organoid acquisition and culture and describe two sample mounting variants for use with multiview and inverted light sheet microscopes.