<p>Conducting toxicological risk assessment of agrochemicals for human health and safety lacks human data and is predominantly based on experimental data obtained from in vivo animal studies. In alignment with the 3R approach (reduce, refine, replace animal testing) and in support of advancing next generation risk assessment of pesticides for human safety, there is a growing demand of implementing new approach methodologies. Therefore, the fungicide trifloxystrobin was investigated concerning human exposure and intestinal first-pass metabolism for the first time using intestinal new approach methodologies. Simulated intestinal fluid containing pancreatin, along with an optimized static in vitro human gut-model (Caco-2/HT29-MTX cell barriers), were established to study the radiolabeled fungicide. A single dose of 10&#xa0;µM was applied to the in vitro systems, resembling an estimated exposure to humans upon oral uptake from food and feed crops. Trifloxystrobin underwent mainly phase I metabolism to its carboxylic acid metabolite, before entering the systemic circulation consistent with findings from in vivo studies with rodents. The results agree with published in vivo and in vitro data and indicate that the carboxylic acid metabolite is a primary metabolite formed before hepatic phase II conjugation. Moreover, minimal passage through the in vitro gut barriers, facilitated by extensive metabolism indicated low oral bioavailability in humans. The presented in vitro methodologies provide valuable insights into the interplay between human intestinal metabolism and oral bioavailability, exemplified by the active ingredient trifloxystrobin. Moreover, the opportunity of transferring the models to more complex microphysiological technologies such as organ-on-a-chip systems, represents an additional value of these models regarding future studies on oral absorption, metabolism, and organ interactions in vitro.</p>