Three-dimensional (3D) cell culture systems and organ-on-a-chip technologies provide more physiologically relevant models compared to traditional two-dimensional (2D) cultures. These advanced models better replicate the in vivo microenvironment, enabling more accurate studies of cellular behavior and drug metabolism, while significantly reducing the need for animal testing. The aim of this study was to develop a 3D equine liver model on an organ-on-a-chip platform and evaluate its metabolic capacity by using various CYP substrates and selected substances prohibited in equine sports (e.g. diazepam). Hepatic spheroids were formed with 2,000 cells per spheroid and were integrated into a dynamic microfluidic system to mimic physiological flow conditions. Metabolic experiments were conducted and the generated metabolites were analysed using liquid chromatography coupled with either a triple-quadrupole or a high-resolution mass spectrometer to assess the model’s potential for generating doping-related compounds. This approach supports applications in drug testing and tissue engineering and offers a practical, scalable, and animal-free platform for advanced 3D liver modeling.