The enteric nervous system consists of a dense, complex network of neurons and glia which are instrumental in the maturation of normal intestinal physiology after birth. Our lab uses a comparative pig model to study the postnatal development of the enteric glial network and its role in regulating intestinal barrier functions in neonates. Immunolabeling‐enabled three‐dimensional (3D) imaging of solvent‐cleared organs (iDISCO) is a method of preparing tissue samples for volume imaging with a light sheet microscope. iDISCO has been optimized for use in primarily mouse organs and embryos for the study of early development and offers a more complete picture of the tissue than traditional histological analysis. Our objective was to optimize the iDISCO protocol for use in porcine intestinal tissue to allow complete qualitative and quantitative analysis of postnatal development of the enteric glial network in our comparative pig model. Antibodies against glial cell markers S100b, Sox10, and glial fibrillary acidic protein (GFAP) were used to triple‐stain fixed full‐thickness 3mm by 5mm samples of porcine jejunum using the iDISCO protocol. Samples were imaged with a light‐sheet microscope (Ultra‐II, LaVision BioTec®) using three different fluorescent channels and datasets were visualized and analyzed in 3D with Imaris software (Oxford Instruments®). The percent volume of GFAP+ glial cells was quantified by manually masking individual intestinal villi and optimizing a surface algorithm to identify glial network structures within those villi. Antibodies against all three markers tested produced the predicted staining pattern with minimal non‐specific staining. Percent of jejunal villus volume occupied by GFAP+ glia is higher in 6‐week‐old versus 2‐week‐old pig (0.49% versus 0.23%, *P≤0.05). Ongoing work will optimize quantification techniques for S100b and Sox10, and assess co‐localization patterns of these glial markers in the jejunum at discrete timepoints postnatally. iDISCO is a powerful imaging modality which will allow our lab to directly assess the expansion, complexity, and localization of glial cell subtypes by marker co‐expression analysis at discrete postnatal timepoints, and will be utilized in future studies to explore effects of disease and external interventions on the enteric glial network.