In the present study, a newly developed nanosecond pulse laser-assisted photoporation using titanium-oxide nanotubes (TNT) for highly efficient intracellular delivery has been established. The proof of concept for the possibilities of intracellular delivery after irradiation of nanosecond pulse laser on TNT has been validated. TNT on titanium sheets using the electrochemical anodization technique at different voltage and time has been developed. The extensive X-ray photoelectron spectroscopy (XPS) study confirms the presence of different titanium oxide species such as TiO2, TixOy (TiO/Ti2O3/Ti3O5) having different concentrations in TNT formed by different anodization voltage and time along with a minor quantity of Ti metal (Ti0). Formation of sub-oxides results in oxygen defects in TNT. It has also been evidenced from XPS that the anodization voltage and time can change the concentration of oxygen defects on the nanotubes. Due to the formation of oxygen defects, nanotubes have the quasi-metallic and metallic properties. These properties of the nanotubes may facilitate the intracellular delivery by various mechanisms after irradiation of nanosecond pulse laser. Using this technique, we successfully have delivered Propidium iodide (PI) and dextran into HeLa cells (HeLa- human cervical cancer cells) with high transfection efficiency and cell viability on nanotubes formed at 15 V/2 h. © 2020