Abstract Background The goal of this study was to understand prospective cohort study Principal Investigators’ (PIs’) attitudes regarding the importance of religion and spirituality (R/S) on disease etiology in order to identify barriers and opportunities for greater inclusion of these domains in high-quality epidemiological research. Methods One-hour, semi-structured qualitative interviews were conducted with 20 PIs, who represent 24 different National Institutes of Health (NIH)-funded prospective cohort studies in the U.S. Collectively, these PIs collect detailed health data on approximately 1.25 of every 100 adult Americans. Sample size was calculated to achieve thematic saturation. Results The majority of PIs we interviewed viewed R/S as potentially important factors influencing disease etiology, particularly among minority communities that report higher levels of religiosity. Yet nearly all PIs interviewed felt there was not yet a compelling body of evidence elucidating R/S influences on health, and the potential mechanisms through which R/S may be operating to affect health outcomes. PIs identified 5 key areas that would need to be addressed before they would be persuaded to collect more R/S measures in their cohorts: (1) high-quality, prospective studies that include all appropriate covariates for the outcome under study; (2) studies that posit a plausible biological mechanism of effect; (3) well-validated R/S measures, collected in common across multiple cohorts; (4) the need to address bias against R/S research among investigators; and (5) NIH funding for R/S research. Conclusions Results of this study provide a roadmap for future R/S research investigating the impact of R/S influences on disease etiology within the context of U.S. prospective cohort studies. Identifying significant R/S influences on health could inform novel interventions to improve population health. Given the higher levels of religiosity/spirituality among minority communities, R/S research may also provide new leverage points for reducing health disparities.