Introduction: Burnout syndrome (BOS) among healthcare workers (HCWs) is a widespread phenomenon that leads to poor staff health and diminished quality of medical care. Spirituality has been proposed as a coping strategy against BOS. The relationship between spirituality and BOS needs to be evaluated. Therefore, a systematic review was conducted to synthesize evidence for understanding association between spirituality and BOS. Methods: The PRISMA guidelines were used to conduct a systematic search of bibliographical databases, including PubMed/Medline, Scopus, and several others in June 2022. Titles. Abstracts, and full-texts of all articles were screened to determine eligibility. The National Heart, Lung, and Blood Institute (NHLBI) quality assessment tool was utilized for the risk of bias (RoB) assessment. Pertinent data were collected using a standardized codebook. Results: Of the original 217 studies, 26 were included in the systematic review, with a total sample of 8,666 participants. Only five studies were interventional or experimental, while the remaining were cross-sectional. Most studies indicated a positive relationship between spiritual well-being or spirituality and personal accomplishment, and a negative association with emotional exhaustion and depersonalization. Interventional studies found that spirituality-based interventions were effective in reducing BOS among HCWs, however, the evidence is fairly scarce. Discussion and Conclusions: There is weak evidence that spirituality is inversely associated with BOS. There are insufficient evidence that spiritual-based health promotion programs in the workplace may be effective in preventing and/or reducing BOS in HCWs.