Stem cells are of outstanding interest for a variety of applications in regenerative medicine. The identification and characterization of novel tissue sources in order to reduce donor site morbidity and to provide specific cells in clinically applicable numbers have led to the detection of stem cells in almost all adult tissues. Salivary glands are of specific interest to our lab, as these tissues are easily accessible for the head and neck surgeon with low donor site morbidity. On the other hand, they possess an endocrine and exocrine function and thus play a very specific role in the human body. Stem cell identity however can only be demonstrated using a combination of different methods in vitro, as there is not a single marker or feature allowing for definite identification of such cells. In this chapter, we provide a comprehensive summary of our experimental methods for the isolation and characterization of human salivary gland stem cells in vitro.