The aim of this research was to develop a method to quantitate the changes in regional brain metabolism on serial FDG-PET scans in children with Traumatic Brain Injury (TBI) in order to determine their response to treatment with psycho stimulant medication. Methods: A Fusion-Based Image Subtraction (FBIS) technique was developed in order to calculate the differences in metabolism between baseline and follow-up scans. The FDG-PET brain images were count normalized to account for differences in the injected activity. Image registration was then performed using a 3D normalized mutual information algorithm and subsequently the fused images were subtracted on a pixel by pixel basis. FBIS extracted areas of increased or decreased metabolism were then compared to a database of normal subjects using NeuroQ (Syntermed, Inc.) and analyzed for statistical significance in cluster areas defined by a standard brain template. Results: Validation of the technique was performed using images from 15 pediatric cases with severe TBI who had baseline (pre-treatment) and follow-up (post-treatment) scans. The FBIS technique was found to be accurate in detecting and localizing the statistically significant changes in regional cerebral metabolism and provided useful data that could be applied clinically. Conclusions: The developed FBIS method identified and quantified areas of regional brain metabolic change thus providing a way to evaluate the effects of pharmacologic treatment in children with TBI. This quantitative technique could be used as an adjunct tool to enhance the interpretation of FDG-PET brain images and aid the referring clinician to assess the response to pharmacologic treatment.