Prior studies addressed the importance of a valid and thorough quantitative instrument for measuring spirituality in a secularized context. However, questions about meaning and spirituality are often limited or absent in multidimensional questionnaires. For that reason, a questionnaire was developed in the Dutch health care setting: Spirituality and Pastoral care In Relation to chronic Illness and Terminal disease (SPIRIT). It is used as a screening instrument in health care practice to identify patients with a chronic or life-threatening disease, who have existential issues. In this article, we present the SPIRIT questionnaire and discuss its reliability and construct validity for screening the spiritual dimension of patients with a chronic or life-threatening disease. We performed two cross-sectional studies (N = 104). Cronbach’s α was used to test the internal consistency of the questionnaire and its scales. Pearson’s correlation coefficient was used to measure the strength and direction of association between variables. Four other instruments were included, covering different aspects of well-being and coping. Most SPIRIT subscales showed an acceptable internal consistency, except for the subscale experiential. Weak and moderate correlations were found between SPIRIT (subscales) and the other instruments. This first, small study to determine reliability and construct validity of the SPIRIT shows it to be a promising screening tool. We expect that the SPIRIT questionnaire aids patients and health care providers to initiate a conversation about this dimension and to determine appropriate follow-up by a spiritual caregiver or other health care professional.