The use of infliximab has completely changed the therapeutic landscape in inflammatory bowel disease. However, despite its proven efficacy to induce and maintain clinical remission, increasing evidence suggests that treatment failure may be associated with inadequate drug blood concentrations. The introduction of biosensors based on different nanostructured materials for the rapid quantification of drugs has been proposed for therapeutic drug monitoring. This study aimed to apply atomic force microscopy (AFM)-based nanoassay for the measurement of infliximab concentration in serum samples of healthy donors and pediatric IBD patients. This assay measured the height signal variation of a nanostructured gold surface covered with a self-assembled monolayer of alkanethiols. Inside this monolayer, we embedded the DNA conjugated with a tumor necrosis factor able to recognize the drug. The system was initially fine-tuned by testing known infliximab concentrations (0, 20, 30, 40, and 50 nM) in buffer and then spiking the same concentrations of infliximab into the sera of healthy donors, followed by testing pediatric IBD patients. A good correlation between height variation and drug concentration was found in the buffer in both healthy donors and pediatric IBD patients (