The cell motility dynamic of two glioblastoma cell lines (U373 and U87) was studied by means of an automatic video-cell-tracking-system enabling each cell in a colony to be tracked for several hours. Twenty-five experiments were performed on both models growing on three different supports (glass, plastic and Matrigel). Cell motility was significantly different in each cell line and also for different growth support in a given cell line. We observed that U87 cells are significantly (p < 0.00001) less motile than U373 cells. The most favorable growth supports for cell motility studies were Matrigel and glass. A significant (p < 0.001) correlation between cell colony density and cell motility was highlighted, with isolated cells exhibiting a motility level distinct from the one observed for colonies. The present methodology, which enabled cell motility to be quantified in human glioblastoma cells, represents an original tool for identifying new classes of compounds able to reduce glioblastoma cell motility and cell migration potential into the brain.