Organoids are three-dimensional (3D) multicellular structures derived from stem cells that recapitulate the architecture and partial functions of their organ of origin. While human organoids are well established across various tissues, the availability of animal-derived organoids remains limited, particularly for respiratory tissues. Given the crucial role of the nasal microbiota as the first line of defense against respiratory pathogens, there is an urgent need for representative in vitro models to study this niche. Here, we present a detailed protocol for the establishment of Porcine Nasal Organoids (PNOs), which enables the investigation of microbial interactions within the porcine nasal cavity in a physiologically relevant context. Furthermore, this model aligns with the 3Rs principles by reducing the need for live animal experimentation.