TiO2-based nanotubes are a very promising material with many applications in solar cells, biomedical devices, gas sensors, hydrogen generation, supercapacitors, and lithium batteries, among others. Nanotube thickness is a very important property since it is related to electronic and surface mechanics. In this sense, transmission electron microscopy (TEM) can be used. However, it can be difficult to acquire a good TEM image because the transversal section of the nanotubes needs to be visible. In this work, TiO2-based nanotubes obtained via hydrothermal synthesis were studied using X-ray line profile analysis. Scherrer and Single-Line methods provided consistent results for the thickness of the nanotubes (≃ 5 nm) when compared with TEM. Additionally, Single-Line method was also applied to estimate the microstrain. The advantage of using XRD is given by the fact that it is a quick and statistically significant analysis when compared with TEM. The results show that XRD can be used as a rapid and reliable alternative for the thickness estimation of nanotubes.