This study aimed to assess the in vitro biocompatibility of titanium (Ti) alloy orthodontic mini-implants by correlating human osteoblasts (HOb) response with chemical composition and surface morphology of mini-implants. HOb were cultivated with or without custom-made and commercial mini-implants, discs and filings. The surface morphology and chemical composition of the implants were assessed under the scanning electron microscopy (SEM) with energy-dispersive X-ray (EDX) microanalysis system. Cell viability, adhesion and proliferation were analyzed by optical microscopy and flow cytometry. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) reduction and lactate dehydrogenase (LDH) release tests were used to assess the cytotoxicity of discs and filings-treated culture medium. Shape, adhesion, and multiplication of HOb were not significantly altered by the presence of mini-implants, discs or filings in culture, even though Ti alloy may exert in vitro a low cytotoxic effect on HOb adhered to discs. Morphology analysis by SEM demonstrated that custom-made mini-implants' surface differs from that of commercial mini-screws in terms of surface finish and roughness, whilst EDX analysis showed largely similar percentages of Ti, aluminum and vanadium for the two types of implants. No major differences were noticed regarding the effect exerted in vitro on HOb by the investigated implants. The new mini-implants have a convenient in vitro cytotoxicity profile on HOb.