Objective: Cotinine is the preferred biomarker to validate levels of tobacco smoke exposure (TSE) in children. Compared to enzyme-linked immunosorbent assay methods (ELISA) for quantifying cotinine in saliva, the use of liquid chromatography tandem mass spectrometry (LC-MS/MS) has higher sensitivity and specificity to measure very low levels of TSE. We sought to compare LC-MS/MS and ELISA measures of cotinine in saliva samples from children overall and the associations of these measures with demographics and TSE patterns. Method: Participants were nonsmoking children (N = 218; age mean (SD) = 6.1 (5.1) years) presenting to a pediatric emergency department. Saliva samples were analyzed for cotinine using both LC-MS/MS and ELISA. Limit of quantitation (LOQ) for LC-MS/MS and ELISA was 0.1 ng/mL and 0.15 ng/mL, respectively. Results: Intraclass correlations (ICC) across methods = 0.884 and was consistent in sex and age subgroups. The geometric mean (GeoM) of LC-MS/MS = 4.1 (range: < LOQ to 382 ng/mL; 3% < LOQ) which was lower (p < 0.0001) than the ELISA GeoM = 5.7 (range: < LOQ to 364 ng/mL; 5% < LOQ). Similar associations of cotinine concentrations with age (β < −0.10, p < 0.0001), demographic characteristics (e.g., income), and number of cigarettes smoked by caregiver (β > 0.07, p < 0.0001) were found regardless of cotinine detection method; however, cotinine associations with sex and race/ethnicity were only found to be significant in models using LC-MS/MS-derived cotinine. Conclusions: Utilizing LC-MS/MS-based cotinine, associations of cotinine with sex and race/ethnicity of child were revealed that were not detectable using ELISA-based cotinine, demonstrating the benefits of utilizing the more sensitive LC-MS/MS assay for cotinine measurement when detecting low levels of TSE in children.includes exposure from both secondhand smoke (SHS) and thirdhand smoke (THS), measurement needs to be reliable, highly sensitive and specific so that even low levels of THS exposure can be detected [1,7]. Nicotine is predominantly derived from combustible and noncombustible tobacco/nicotine products. Over 70% of nicotine is metabolized to cotinine by the liver enzyme CYP2A6 [8]. Cotinine is nicotine's major proximate metabolite with a mean half-life of 16 h; some studies have reported higher median half-life levels of up to 28.3 h in infants and older children [1,[9][10][11]. In combination with cotinine, the other nicotine metabolites are nicotine, trans-3'-hydroxy cotinine (3HC), cotinine glucuronide, nicotine glucuronide, and 3HC glucuronide, which in combination, estimates the approximate daily intake of nicotine [1,12]. Cotinine concentrations are relatively stable and can be detected in the blood, saliva, urine, hair, and nails of children who are exposed to SHS and THS [1]. Thus, cotinine is the most commonly reported measure to indicate children's recent exposure to tobacco smoke. Since the concentration of cotinine in blood and saliva are highly correlated, saliva is often used to assess TSE in children since the collect...