A reliable quantification technique for interpreting nanomaterial-substrate bond strength is highly desired to predict efficient, long-term performance of nanomaterial-based devices. Adopting a novel nanoscratch-based technique, here we demonstrate quantification of carbon nanotube (CNT)-substrate adhesion strength for dense CNT structure and for patterned carbon nanocone (CNC) structures. Debonding energy for a single CNT is illustrated to range between 1 and 10 pJ, and the variation is strongly dependent on the nature of the interface between CNTs, catalysts, and substrates. Our proposed technique could be adopted for characterization of bonding strength between a wide variety of nanotubes, nanowires, and other one-dimensional nanostructured materials and their underlying substrates.