Laboratory detection of nicotine exposure is important for establishing eligibility for organ transplant and elective surgery. Nicotine testing is also used to verify compliance with nicotine replacement therapies (NRT), smoking cessation programs and for life insurance purposes. Nicotine metabolites, such as cotinine and trans-3'-hydroxycotinine, are used as biomarkers of nicotine exposure. For some clinical applications, it is important to distinguish between active use of tobacco products versus NRT. Anabasine is a tobacco alkaloid that has been used as a biomarker of active tobacco use. However, the use of anabasine as an insecticide, and its presence in consumables other than nicotine products, suggests that anabasine may not be specific to tobacco use/exposure. Here, we determine the reference interval for anabasine in the urine of nonsmokers and compare it to the range of anabasine concentrations observed in the presence or absence of nicotine metabolites.