The involvement of the Enteric Nervous System (ENS), the gut’s brain, in digestive and neurological disorders is widely recognized and constitutes a novel target for diagnostic biomarkers and therapies. For example, Parkinson’s disease patients are now stratified according to gut-first or brain-first patterns that diverge in symptom progression. However, large-scale characterization of this neuronal network is often limited to dissected micrometer-scale samples that cannot reflect network changes. We present a method for 3D quantitative analysis of ENS morphology using fluorescence lightsheet microscopy in rodent and human tissues. Spanning from sample collection to data analysis, this method takes up to 3 weeks to produce data in multiple cubic centimeters of tissue at near-cellular resolution. Parameters such as network integrity and connectivity can be observed, quantified and used to assess ENS health status. Furthermore, we demonstrate label-free segmentation of the ENS thanks to endogenous autofluorescence signals, cutting down processing time to 3 days and making it a viable option for rapid ENS characterization in biopsies. The developed pipeline will be applied in both healthy and Parkinson’s disease model animals with the aim to compare gastrointestinal and enteric network morphology. With this method, we hope to enable more research to span from the brain to the gut and to shed light on the potential of monitoring ENS remodeling as a diagnostic tool for patient management.