Metabolomics makes it possible to analyze the interrelationships between various signaling molecules based on the metabolic pathways involved by using high-resolution devices. This approach can also be used to obtain large-scale metabolic information to identify the relevant pathways for disease diagnosis and prognosis and search for potential biomarkers. In the fields of medicine and forensics, hair analysis is used to detect various metabolites in the body. Hair can be harvested readily in a noninvasive manner and is easier to transport and store than blood and urine. Another advantage from a forensic viewpoint is that hair reflects all the components of body fluids. In addition, because of the unique coating structure of hair, it can be used for measurements without changing or destroying its adsorbed components. In this review, the pretreatments for hair analysis, instrumental conditions and clinical applications are discussed. Especially, the clinical use of hair metabolomics in the diagnosis of various diseases and the limitations of the technique are described.