Hydroxycotinine is primarily used as a biomarker in medical and research settings to assess tobacco exposure and nicotine metabolism. It is a major metabolite of cotinine, which itself is a metabolite of nicotine, making it a reliable indicator for detecting and quantifying tobacco use. In clinical studies, hydroxycotinine levels are measured in blood, urine, or saliva to evaluate smoking habits, monitor smoking cessation programs, and study the metabolic pathways of nicotine. Additionally, it helps in understanding individual variations in nicotine metabolism, which can influence addiction patterns and the effectiveness of nicotine replacement therapies. Its application extends to forensic science, where it is used to confirm tobacco use in legal or workplace testing scenarios.