The application of nanomaterials in the fields of medicine and biotechnology is of enormous interest, particularly in the areas where traditional solutions have failed. Unfortunately, there is very little information on how to optimize the preparation of nanomaterials for their use in cell culture and on the effects that these can trigger on standard cellular systems. These data are pivotal in nanobiotechnology for the development of different applications and to evaluate/compare the cytotoxicity among the different nanomaterials or studies. The lack of information drives many laboratories to waste resources performing redundant comparative tests that often lead to partial answers due to differences in (i) the nature of the start-up material, (ii) the preparation, (iii) functionalization, (iv) resuspension, (v) the stability/dose of the nanomaterial, etc. These variations in addition to the different analytical systems contribute to the artefactual interpretation of the effects of nanomaterials and to inconsistent conclusions between different laboratories. Here, we present a brief review of a wide range of nanomaterials (nanotubes, various nanoparticles, graphene oxide, and liposomes) with HeLa cells as a reference cellular system. These human cells, widely used as cellular models for many studies, represent a reference system for comparative studies between different nanomaterials or conditions and, in the last term, between different laboratories.