Description: Benzo[a]pyrene (BaP), is a human carcinogen and is formed during the incomplete combustion of organic matter, like tobacco smoke. Urinary 3-hydroxybenzo[a]pyrene (3-OH-BaP) can be assessed as biomarker of BaP exposure. We developed and validated a sensitive method for the quantification of 3-OH-BaP to investigate the contribution of smoking and use of potentially reduced risk products to the exposure to BaP in non-occupationally exposed subjects. Application of the method to urine samples from a controlled clinical trial showed that urinary 3-OH-BaP concentrations were significantly higher in smokers compared to users of potentially reduced risk products as well as non-users. Due to its high sensitivity, accuracy, and precision, this method is well suited to distinguish cigarette smokers from other nicotine consumer groups in non-occupationally exposed populations.