<p>Enniatin B1 (EnnB1) is a <i>Fusarium</i>-produced mycotoxin contaminating cereals and cereal-based products, which represent the main source of human exposure to the toxin. EnnB1 belongs to the “emerging mycotoxins” group that are not regulated due to data gaps on their toxicokinetic and toxicodynamic profiles. The aim of this study was to determine the in vitro hepatic clearance of EnnB1 based on parent compound depletion. To do this, we used two in vitro experimental assays—a conventional and a media loss assay—and compared hepatic clearance between (i) species, using primary rat and human hepatocytes, (ii) cell types, namely primary human hepatocytes and the HepaRG human hepatic cell line, and (iii) cell models by comparing 2D and 3D HepaRG. Hepatic clearance (Cl_H) was calculated from in vitro hepatic intrinsic clearance using in vitro to in vivo extrapolation. We found that the derived Cl_H was faster in rats compared to humans, i.e., 3.4 L/(h·kg) and 1 L/(h·kg), respectively. Cl_H was comparable between primary human hepatocytes and HepaRG 2D, while HepaRG 3D showed slower Cl_H of 0.58 L/(h·kg). We found that the media loss depletion assay revealed an initial phase of rapid uptake, then a slope parallel to that of the conventional depletion assay. We observed that the conventional depletion assay was appropriate to determine the Cl_H of EnnB1. Furthermore, the pooled primary hepatocytes, considering inter-individual variability, allowed the determination of a reliable Cl_H to be used in the development of a physiologically based pharmacokinetic model.</p>