The liver is the principal detoxification center of the body, removing xenobiotics and waste products which could potentially include some nanomaterials (NM). With the ever increasing public and occupational exposure associated with accumulative production of nanomaterials, there is an urgent need to consider the possibility of harmful health consequences of engineered NM exposure. It is understood that following exposure via inhalation, ingestion, or direct intravenous injection a fraction of NMs reach the liver. Traditional in vitro or ex vivo hepatic nanotoxicology models are often limiting and/or troublesome (i.e., reduced metabolism enzymes, lacking important cell populations, unstable with very high variability, etc.). This chapter highlights a methodology for the preparation of a physiologically relevant 3D human liver microtissue model which addresses most of the negative issues associated with the models used in traditional in vitro hepatic toxicological investigations. The spheroids are a very promising model for the assessment of the toxicological effects associated with engineered NM exposure.