In the last 30 years the research about zinc oxide nanoparticles (ZnO NPs) and their related toxicity has shown a boom. ZnO NPs show cytotoxicity for both prokaryotic and eukaryotic cells and many studies demonstrated their selective toxicity towards cancer cells. However, with the increasing number of publications, it is observed an increase in the discrepancies obtained between the various results. Soon the scientific community understood that the ZnO NC toxicity behaviour is affected by many factors, related not only to the ZnO NPs themselves, but also to the experimental conditions used. Many recent reviews discussed these parameters by reporting experimental evidence and tried to assess the general statements about the ZnO NP cytotoxicity. This information is extremely useful for the evaluation of which type of ZnO NPs is more or less suitable for a specific study or application. However, despite that, a deep comprehension of the ZnO NP behaviour in relation to the different experimental conditions is still lacking. Actually, a full understanding of the reasons behind the NP behaviour is essential to better assess their biological activity and in particular their therapeutic application, avoiding undesired effects both in the experimental and clinical contexts. This tutorial review aims to be an experimental and practical guide for scientists that faced with the use of ZnO NPs for biomedical applications and, in particular, for their therapeutic purposes. The driving idea is to not simply summarize the results reported in the literature, but to provide instruments for a deep comprehension of the mechanisms affecting the ZnO NP cytotoxicity and behavior. This review also aims to point out the critical experimental parameters to be considered when working with these NPs, as well as the main related risks and limitations that scientists have to face.