Background <p>Hirschsprung disease (HD) is a congenital malformation characterized by the absence of ganglion cells in the bowel wall. The aganglionosis extends from the rectum and proximally into the gastrointestinal tract in various lengths. The diagnosis relies on histological examination of rectal biopsies. Two main biopsy techniques: rectal suction biopsy (RSB) or full-thickness biopsy (FTB) are available. They differ in the degree of invasiveness, postoperative complications and diagnostic accuracy.</p> Methods <p>This is a prospective trial including 300 patients aged 0–17&#xa0;years scheduled for rectal biopsy on the suspicion of HD. Each patient will undergo both RSB and FTB and thus serve as its own control. The sequence of histological analysis of RSB and FTB will be randomized. The primary objective is to compare the diagnostic performance of FTB and RSB in children evaluated for Hirschsprung’s disease. Specifically, the aim is to assess how effectively each technique obtains adequate tissue samples for nerve cell evaluation and to compare the rates of inconclusive biopsies associated with each method. The secondary objective is to evaluate the concordance to the National Institute for Health and Care Excellence (NICE) symptom-based criteria for rectal biopsy in children with severe constipation.</p> Discussion <p>The findings from the BIOPSY-STUDY are expected to provide high-quality evidence to guide biopsy selection in suspected Hirschsprung’s disease. By directly comparing RSB and FTB within the same patients, the study aims to clarify diagnostic accuracy, reduce inconclusive results, and support more efficient, cost-effective clinical pathways. Despite being single-center, the rigorous design enhances internal validity and clinical relevance.</p> Trial registration <p>The study is registered at ClinicalTrials.gov NCT05307419. Registered on April 01, 2022.</p>