The detrimental effects of stress on human health are being increasingly recognized. There is a critical need for the establishment of a biomarker that accurately measures its intensity and course over time. Such a biomarker would allow monitoring of stress, increase understanding of its pathophysiology and may help identify appropriate and successful management strategies. Whereas saliva and urine cortisol capture real-time levels, hair cortisol analysis presents a complementary means of monitoring stress, capturing systemic cortisol exposure over longer periods of time. This novel approach for cortisol quantification is being increasingly used to identify the effects of stress in a variety of pathological situations, from chronic pain to acute myocardial infarctions. Because of its ability to provide a long-term, month-by-month measure of systemic cortisol exposure, hair cortisol analysis is becoming a useful tool, capable of answering clinical questions that could previously not be answered by other tests. In this paper we review the development, current status, limitations and outstanding questions regarding the use of hair cortisol as a biomarker of chronic stress.