The greatest disease-producing product known to man is tobacco. It is a cause of many oral diseases and adverse oral conditions. In India, tobacco is available in smokeless and smoking form. Tobacco contains nicotine which metabolises to form a toxic alkaloid i.e. cotinine. It stimulates autonomic ganglia and central nervous system. Cotinine is the best indicator of tobacco smoke exposure. Various methods are used to measure cotinine level in blood, saliva and urine such as high performance liquid chromatography, colorimetric assay, gas chromatography, NicAlert saliva test, etc. Thus such wide range of methods for cotinine detection in tobacco users requires a detailed discussion regarding their utility. This review will help readers to compare various methods for cotinine detection and enable them to make scientifically informative decision.