Glioblastoma multiforme (GBM) is a highly invasive (WHO grade IV) brain tumour that has a very poor prognosis for patients with the condition (median survival 14.2 months). Quantitative Imaging (QI)® mode atomic force microscopy (AFM) was used to measure the heights of the leading-edge cell peripheries, the lamellipodia, of two such cell lines (SNB-19 and UP-007), together with those from non-neoplastic astrocyte control cells (CC-2565 and SC-1800) and from a low-grade (WHO grade I) glioma cell line (SEBTA-048). The lamellipodia heights of the glioma cells SNB-19 and UP-007 were 2.45 ± 0.59 and 1.57 ± 0.42 μm, respectively, which were higher than those of the CC-2565 and SC-1800 cells (1.03 ± 0.58 and 0.85 ± 0.40 μm, respectively; p < 0.0001, except between CC-2565 and UP-007, p < 0.001). Lamellipodia height differences between the two glioma cell lines (p < 0.0001) might be attributed to the measured difference in invasive potential between these two cell lines. The equivalent lamellipodia height of the SEBTA-048 cells was 1.16 ± 0.48 μm, the same as that of the astrocytes (p > 0.05) but lower than those of the high-grade gliomas (p < 0.0001 and p < 0.01 for SNB-19 and UP-007, respectively). These measured heights, therefore, may provide new insights for monitoring and controlling cellular invasion in brain tumours.