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Nanoparticle (NP)-based solutions for oncotherapy promise an improved efficiency of the anticancer response, as well as higher comfort for the patient. The current advancements in cancer treatment based on nanotechnology exploit the ability of these systems to pass biological barriers to target the tumor cell, as well as tumor cell organelles. In particular, iron oxide NPs are being clinically employed in oncological management due to this ability. When designing an efficient anti-cancer therapy based on NPs, it is important to know and to modulate the phenomena which take place during the interaction of the NPs with the tumor cells, as well as the normal tissues. In this regard, our review is focused on highlighting different approaches to studying the internalization patterns of iron oxide NPs in simple and complex 2D and 3D in vitro cell models, as well as in living tissues, in order to investigate the functionality of an NP-based treatment.