Gastric cancer is an important healthcare problem and represents the second leading cause of death for malignant disease worldwide. In the Western world, the diagnosis is done at late stage when treatments can be only palliative. Searches for new therapeutic regimens as well as for new biomarkers are in progress. To reduce cancer mortality is crucial the prevention of the lesion at earlier stages. Therefore, new bullets to prevention are needed. Nowadays, studies relating to different kinds of tumor are unanimous in considering cancer stem cells (CSCs) as "the core" of the tumor and the responsible of tumor chemoresistance and relapse. This chapter aims to provide the instructions to (1) isolate, (2) grow, and (3) validate, both in vivo and in vitro, the gastric CSC subpopulation.