Background and Objective: A thorough study was designed with an objective of observing utility of calretinin as immune-histochemical marker for aganglionosis and for detection of ganglion cells in the inadequate biopies of affected areas for more accurate and better diagnosis of the disease. Study Design: It was observational, descriptive study. Setting: It was carried out at the Department of Morbid Anatomy and Histopathology in University of Health Sciences (UHS) Lahore. Period: The study commenced in March 2016 after approval of the synopsis by the Advance Studies and Research Board of UHS and was successfully completed in December 2016. Methodology: Biopsy specimens of colon which were considered for the study were collected from 73 patients from Mayo Hospital and Jinnah Hospital, Lahore with established histopathologically diagnosed HSCR on H&E staining. Results: The mean age was 12.52 ± 9.21 months. On the basis of Calretinin staining Ganglion cells were present in 42/73 (57.53%) and absent in 31/73 (42.47%) respectively. Conclusion: It was consummated that Calretinin provides a reliable and very cost effective adjunctive test to be used routinely with H&E in diagnosing HSCR and consequently waiving off the need for unnecessary surgeries and repeated biopsies.