Glioblastoma tumors form in brains' white matter and are fast-growing and aggressive. Poor prognosis is the result of therapeutic resistance and infiltrating growth into the surrounding brain. Here we present a protocol for the detection of the cytoskeleton intermediate filament, vimentin, in cells at the proliferating spheroid surface. By combining a classical invasion assay with immunofluorescence and light-sheet imaging, we find that it is exactly these cytoskeleton-reinforcing cells on the spheroid's surface that will start the infiltration. We anticipate our results to be the starting point of more sophisticated investigation of anti-cancer drug effects on cytoskeleton reorganisation.