Glioblastoma multiforme (GBM) is the most common and biologically aggressive brain tumour. The current standard therapy for GBM consists in surgical resection, followed by radiotherapy and chemotherapy. Yet, the treatment is limited due to the area for the surgical resection and for the inability of some drugs to cross the brain blood barrier, leading to a general prognostic of no more than a year. Cold atmospheric plasma (CAP) is a new approach in the treatment of this challenging disease. CAP interaction with cells is dependent on physical and chemical factors, with different plasma discharges, cell type, and culture conditions leading to different CAP activity. Considering the plasma self-adaptation that different plasma discharge modes can undergo, which leads to different interaction plasma/cells, the characterization of a new device is essential. In this study we analysed the effect of a novel large pin-to-plate non-thermal atmospheric plasma on U-251 MG cells under different conditions. The analysis of reactive oxygen and nitrogen species (RONS) on plasma, media and cells were also assessed. We were able to demonstrate that the pin-to-plate device is cytotoxic to GBM cells in a dose, time and ROS dependent manner. The measurements of RONS on plasma/media also give us an insight on the chemical effect of this novelty device, and the possibility to better understand the use of this device as a promising GBM therapy.