Throughout the years, the respiratory research community has immensely benefitted from studies using in vitro and in vivo models to either dissect or integrate the contributions of cellular and molecular components across multiple respiratory compartments. The introduction of technology that allows spatial transcriptomic and multiplex protein expression analyses has recently bolstered a new understanding of the value of spatial context. Just like in conventional histopathology, the ability to preserve and capture a particular spatial organization is key to the success of these analyses. This Methods Collection includes validated protocols to harvest, fix, and process samples from in vitro (airway epithelial cells cultured on permeable substrates) and in vivo (various compartments of the murine respiratory tract) models commonly used to investigate respiratory phenotypes.