One of the major research interests of nanomedicine is the designing of harmless and biocompatible medical devices. To improve the features of Ti surface, TiO2 based nanotube (TNT) films (50 nm diameter) achieved by anodic oxidation and thermal treatment were grown on titanium and on Ti6Al4V and Ti6Al7Nb alloys. Their in vitro toxicity and biocompatibility were investigated using G292 osteoblast cell line. The LDH release after 24 and 48 h of exposure demonstrated that TNT layers were not cytotoxic. The cell growth on TNT films deposited on titanium and Ti6Al4V was significantly increased compared with Ti6Al7Nb. F-actin staining showed a better organized actin cytoskeleton in osteoblasts grown on these two samples, which provide the best conditions for osteoblast attachment and spreading. Analysis of GSH distribution revealed a higher nuclear level in the samples with TNTs compared with Ti plate without nanotubes, indicating an active proliferation. Thus, nuclear glutathione levels can be used as a useful biomarker for biocompatibility assessment. Our results suggest that the substrate for TNTs can have a significant impact on cell morphology and fate. In conclusion, the TNT/Ti and TNT/Ti6Al4V were toxicity-free and can provide a proper nanostructure for a positive cell response.