Tobacco smoke exposure is the primary cause of disease and premature death in the U.S. population and a major contributor to negative health impact globally. Tobacco smoke harms the health of active users and non-users with indirect exposure. Biomarkers of exposure to tobacco smoke are crucial for characterizing exposure and evaluating the health impact of exposure, especially as the tobacco product marketplace changes and non-combustible tobacco products are used more widely. The objective of this presentation is to (1) underscore the importance of analytical methods in ensuring the long-term accuracy and precision of reported biomarker data, (2) summarize tobacco-related biomarker data available as part of the NHANES, and (3) highlight the usefulness of combinations of tobacco-related biomarkers for characterizing exposures. The presentation will include examples of the U.S. Centers for Disease Control and Prevention (CDC) quality assurance/quality control (QA/QC) efforts to ensure long-term stability of analytical methods so that exposure trends across decades can be reliably evaluated. Additionally, the availability of tobacco exposure biomarkers in NHANES will be discussed. The presentation will primarily focus on selective smoke exposure biomarkers, including 1-hydroxypyrene, thiocyanate, acrylonitrile metabolite N-acetyl-S-(2-cyanoethyl)-L-cysteine (CYMA), and 2-Amino-9H-pyrido[2,3-b]indole (AaC). These tobacco smoke exposure biomarkers will be compared with cotinine for distinguishing among different types of tobacco product use. For example, sample-weighted median urinary CYMA levels were much higher among exclusive smokers 147 [25^th, 75^th percentile: 69.6, 245] compared with non-users (1.41 [0.93, 2.20] µg/g creatinine). Urinary CYMA is an excellent biomarker of exclusive smoking vs. non-users, as indicated by its sample-weighted area under the ROC curve (AUC; 0.958 [95 percentile: 0.947, 0.969]), and which was comparable to serum cotinine’s (0.970 [0.960, 0.980]). In conclusion, urinary CYMA is an excellent biomarker of smoke exposure, especially in situations where smoke exposure is of primary interest and is to be assessed independently from nicotine exposure.