A study was undertaken on the influence of ultrasonication over the second-generation TiO2 nanotubes structured matrix. A 60 V applied voltage for 2 hours anodizing was used to create the ordered structures, with a high voltage power source in low water glycerol electrolyte. The results were investigated using Environmental Scanning Electron. The ultrasonication was performed after anodizing using an ultrasonic cleaner. In correlation to different ultrasonication times, a variance of nanotubes/nanopores was observed. The atomic composition at the surface indicates proportionality between Ti and O in the nanostructures layers and an increase in fluorine with the increase of ultrasonication time. These measurements give information on the wall composition as nanotubes layers are removed through the ultrasonication process. The surface analysis and roughness evaluation of the Ti/TiO2 nanotube surface were completed with Atomic Force Microscopy (AFM) using an atomic force microscope. The electrochemical behavior of the nanotubes structured surface was performed using electrochemical impedance spectroscopy (EIS).