Up to now, a quantitative three-dimensional (3D) assessment of the lacuno-canalicular network (LCN) within bone has not been achieved in a comprehensive way and the LCN has mostly been investigated using two-dimensional imaging methods only. First attempts for the 3D assessment of the osteocytes and their cell processes have been reported using different imaging techniques. Nevertheless, various experimental limitations allowed for assessment of isolated or incompletely interconnected osteocytes only. On the other hand, serial focused ion beam/scanning electron microscopy (FIB/SEM) currently seems to be a promising imaging method for quantitative 3D assessment of the LCN. However, combined 3D visualization and quantification of the LCN using serial FIB/SEM imaging has not been reported so far. The aim of this study was to provide a proof of concept that serial FIB/SEM meets all requirements for quantitative 3D imaging of the LCN. To this end, we developed a new bone sample preparation protocol for serial FIB/SEM imaging providing a resolution on the order of 30 nm. This technique was successfully applied to the mid-diaphysis of a mouse femur. Moreover, we devised and applied novel measures for subsequent quantitative 3D morphometry of the LCN. Briefly, serial FIB/SEM was shown to be an appropriate technique to quantify the morphology of the LCN truly in 3D. This will allow investigating bone matrix changes on an ultrastructural level, which result from aging, disease, and treatment.