Here, we report the development of a novel in vitro digestion system that mimics the digestive process in fish and uses the RTgutGC cell line, a model of the rainbow trout (Oncorhynchus mykiss) intestine, to study the bioavailability, bioreactivity, and toxicity of Zn added as an inorganic (ZnSO₄) and organic zinc compound (Zn-Bioplex). Our results showed that, before digestion, Zn-Bioplex was more bioavailable than ZnSO₄. However, after in vitro digestion, ZnSO₄ and Zn-Bioplex accumulated and were transported equally across the RTgutGC cell epithelium. However, Zn-Bioplex was shown to be less bioreactive than ZnSO₄, suggesting that peptide complexation might reduce the intracellular release of zinc as shown by a reduced induction of metallothionein and ZnT1 mRNA levels. This novel method shows promise as a standardized in vitro fish digestion model to study the interactions occurring at the lumen of the intestine and the bioavailability and toxicity of digested compounds in the fish gut.