The aim of this study was to evaluate the interspecies variation in APIs’ clearances between commercially available primary fish hepatocytes. For this purpose, six APIs with varying metabolic profiles (in human) were screened for their intrinsic clearances (CLINT) in rainbow trout, Atlantic salmon and common carp, first using hepatocyte suspensions (OECD 319A). The results revealed notable variation between species in terms of both clearance rates and metabolic pathways (assessed based on supplemental assays with liver S9 fractions of the same species), with rainbow trout typically exhibiting the highest clearance rates and common carp the lowest rates.