Hirschsprung's disease (HD) is a congenital intestinal disease that is clinically manifested as an inability to pass meconium in infants or as long-term constipation in children. Rectal suction biopsy (RSB) to determine the absence of ganglion cells and neural hypertrophy is the most accurate test for the diagnosis of HD at present. Traditional hematoxylin-eosin staining lacks sensitivity and specificity. Acetylcholinesterase staining cannot be widely used due to its complex process. Our novel protocol of immunostaining for calretinin, S100 protein, and protein gene product 9.5 (PGP9.5), which we conducted on RSBs, exhibits high sensitivity and specificity rates of 96.49% (95% confidence interval, 0.88-0.99) and 100% (95% confidence interval, 0.97-1.00), respectively. The HD-affected segments often present as the absence of the expression of calretinin, S100 protein, and PGP9.5, which are markers of neural hypertrophy in the submucosal tissue. This protocol describes the detailed operating process of this new diagnostic method.