Amphihevir, a benzofuran derivative, is the first reported hepatitis C virus (HCV) nonstructural protein 4B (NS4B) inhibitor that has advanced to clinical trials (currently in phase Ib trial [CTR20170632]). Here, we report the results of a preclinical study of its potency, toxicity, selectivity, drug metabolism and pharmacokinetics (DMPK), and safety profiles.