Hydroxylated polybrominated diphenyl ethers (OH‐PBDEs) are of particular concern due to their ubiquitous distribution and adverse health effects. Significant progress has been made in the characterization of OH‐PBDEs by using mass spectrometry (MS). In this review, we summarize applications of MS‐based techniques in detection, environmental and biota distribution, and potential health risk effects, hoping to unfold an overall picture on account of current knowledge of OH‐PBDEs. The analytical methodologies are discussed from sample pretreatment to MS analysis. The methods including gas chromatography‐MS (GC‐MS), liquid chromatography‐MS (LC‐MS), and ion mobility spectrometry‐MS (IMS‐MS) are discussed. GC‐MS is the most frequently adopted method in the analysis of OH‐PBDEs due to its excellent chromatographic resolution, high sensitivity, and strong ability for unknown identification. LC‐MS has been widely used for its high sensitivity and capability of direct analysis. As a newly developed technique, IMS‐MS provides high specificity, which greatly facilitates the identification of isomers. OH‐PBDEs pervasively existed in both abiotic and biotic samples, including humans, animals, and environmental matrices. Multiple adverse health effects have been reported, such as thyroid hormone disruption, estrogen effects, and neurotoxicity. The reported potential pathological mechanisms are also reviewed. Additionally, MS‐based metabolomics, lipidomics, and proteomics have been shown as promising tools to unveil the molecular mechanisms of the toxicity of OH‐PBDEs. © 2020 John Wiley & Sons Ltd. Mass Spec Rev