Glioblastoma multiforme (GBM) are the most common malignant brain tumours in adults, characterized by short survival periods of patients. Their aggressive local growth pattern and increased invasiveness, due to a high motility of the tumour cells, hamper treatment. However, the molecular mechanisms regulating glioblastoma cell migration are still elusive. Here, we describe the combination of a highly efficient cell transfection by nucleofection technology and the generation of spheroids from these transfected glioblastoma cell lines. Nucleofection allows the manipulation of protein expression by overexpression and siRNA-mediated protein knock-down. Transfection efficiencies >80% can be achieved with some GBM cell lines. Transfected neurospheres then can be used for migration assays (as described here in detail) and a multitude of other functional assays. In comparison to monolayer cultures, the advantage of spheroids is their resemblance to organized tissue in combination with the accuracy of in vitro methodology and marked experimental flexibility.