Background : The diagnosis of Hirschsprung′s disease (HD) is based on the absence of ganglion cells. In hemotoxilin and eosin (H and E) as well as acetylcholine esterase staining there are limitations in the diagnosis of immature ganglion cells in neonates. Methods : In this prospective study, 54 biopsies taken from suspected HD patients (five mucosal specimens and 49 full thickness specimens) were studied. In the laboratory, after preparing sections of paraffin embedded tissues, H and E staining slides were compared with immunohistochemical (IHC) staining including: S100, NSE, CD117, CD56, Cathepsin D, Vimentin, BCL2, GFAP, Synaptophysin and chromogranin. Results : The study revealed 30 negative (absence of ganglion cells) cases (55.5%), 17 positive cases (31.04%) and seven suspected cases (12.9%) of ganglion cells on the H and E staining. On IHC staining with CD56 and Cathepsin D, all of the 17 positive cases detected through H and E, were confirmed for having ganglion cells and out of 30 cases reported negative on H and E staining, 28(93.3%) were reported negative and two (6.7%) positive by IHC staining. Of the seven suspected cases H and E staining), IHC staining detectedganglion cells only in five slides; two remained negative. Conclusions : IHC staining using CD56 and Cathepsin D improved the accuracy of diagnosis in HD when used in addition to H and E staining technique, especially for negative or suspicious slides.