Herein, we present an ex vivo approach to study glioblastoma (GBM) cell motility in viable mouse brain slice cultures, closely mimicking in vivo features. We detail the preparation and culturing of mouse brain slices followed by tumor cell injection, allowing for the analysis of different aspects of the cellular migration and invasion process. Our assay facilitates testing diverse perturbations including genetic modifications and treatments in a physiological context. Thus, the protocol provides a compromise between in vitro assays and in vivo models. For complete details on the use and execution of this protocol, please refer to Delbrouck et al.¹ and Schuster et al.²