Interstitial Cells originate from mesenchymal embryonic layer communicate with smooth muscle cells in many organs and provide important regulatory functions. Two different classes of Interstitial Cells (ICs) namely, cKit+ and PDGFRα+ ICs have been described in detail in the gut tissue. They represent distinct groups of cells with distinctive ultrastructure. Both cell types are electrically coupled to smooth muscle cells (SMCs) and enteric neuronal cells, together forming an integrated unit called the SIP syncytium. SIP cells express a range of receptors and ion channels such as Ca2+- activated Cl- channel (Ano1) and Ca2+-activated K+ channels (SK3), changes in the conductance of any type of SIP cells affect the excitability of the whole syncytium unit. cKit+ ICs provide pacemaker activity, slow wave propagation pathways and motor neurons input transduction. Loss of ICs has been associated with gut motility disorders. However, the pathophysiological roles of these cells were not clearly defined in most cases. Gastrointestinal smooth muscles produce electrical activity (slow waves) that is not dependent on nerve input. Intrinsic pacemaker activity comes from cKit+ ICs, which are electrically coupled to SMCs via gap junctions. Abnormalities in ICs numbers and networks has been linked to several gastrointestinal motility disorders. Hirschsprung disease (HD) is a congenital malformation of the enteric nervous system (ENS). The diagnosis is generally established by an experienced pathologist based on histopathological studies of rectal biopsies. Obstructive symptoms such as constipation or recurrent intestinal dysmotility problems arecommon after definitive surgery. Defining how ICs are affected in HD allows insights into the interdependence of the ENS and ICs. Furthermore, these post-operative complications have led researchers to consider alternative therapies based on understanding the role of ICs in developing the HD. This project describes different protocols including, immunohistochemistry, in situ hyberdization, qPCR and RNAseq for identifying ICs in a step toward understanding their contributions in gut motility disorder especially HD. However, further experiments and computational techniques are required to confirm the function of ICs in HD.