It has recently been shown that hydroxyl radicals are generated under physiological and pathological conditions and that they seem to be closely linked to various models of pathology putatively implying oxidative stress. It is now recognized that the hydroxyl radical is well-regulated to help maintain homeostasis on the cellular level in normal, healthy tissues. Conversely, it is also known that virtually every disease state involves free radicals, particularly the most reactive hydroxyl radical. However, when hydroxyl radicals are generated in excess or the cellular antioxidant defense is deficient, they can stimulate free radical chain reactions by interacting with proteins, lipids, and nucleic acids causing cellular damage and even diseases. Therefore, a confident analytical approach is needed to ascertain the importance of hydroxyl radicals in biological systems. In this paper, we provide information on hydroxyl radical trapping and detection methods, including liquid chromatography with electrochemical detection and mass spectrometry, gas chromatography with mass spectrometry, capillary electrophoresis, electron spin resonance and chemiluminescence. In addition, the relationships between diseases and the hydroxyl radical in living systems, as well as novel separation methods for the hydroxyl radical are discussed in this paper.