Cortisol is the primary glucocorticoid produced by the activation of the hypothalamic pituitary adrenal (HPA) axis after a psychological or physiological stressor. The dysregulation of the HPA axis by chronic stress has been associated with psychiatric disorders. Although hair is currently the main validated source of chronic cortisol concentrations, cortisol is also bound to human nails, another keratinised matrix. Therefore, nail cortisol has the potential to be an alternative retrospective chronic measure of HPA activation. The aim of this systematic review was to assess the temporal resolution, methodological issues, HPA correlates, and target populations in nail cortisol investigations. A qualitative synthesis was performed to assess current literature exploring cortisol concentrations from human nails. A total of 18 eligible human studies extracted from Medline (PubMed and Ovid), ProQuest (PsycINFO), and Scopus found that immunoassays and mass spectrometry were the two primarily methods of analysis. However, methodological variability remained evident between studies. Nail cortisol correlated with saliva and hair in some studies and was investigated across multiple developmental periods. Finally, when applied as an outcome measure in health disorders, higher nail cortisol concentrations have been shown to be associated with acute coronary syndrome and depression. In conclusion, nail cortisol may serve as a retrospective biomarker of chronic stress; however, the ability to track how much cortisol is accumulating within nail clippings is complex and may represent a large timespan. Further, very few studies have reported effect sizes and investigated the effects of covariates, such as age, sex, ethnicity, and nail characteristics, which limits the validation of this measure. Further studies are required to validate the utility of nail cortisol as a biomarker of chronic stress across the human lifespan.