Glioblastoma is the most aggressive and the most common type of brain malignant tumors in adults with an average survival of 12 months after diagnosis. This poor prognosis is due to the fast growth and highly infiltrative feature of glioma cells, which are able to migrate from the main tumor mass and invade the normal brain parenchyma [1–4]. Preoperative imaging does not always clearly define the edge of the tumor because of this infiltration, and the presence of a positive margin after surgery contributes to frequent recurrence rates . In this study a new contrast agent consisting of a fluorescently labeled Affibody molecule that binds to the epidermal growth factor receptor (EGFR) was examined for uptake in orthotopic glioma tumors to assess the contrast available to guide surgery.