AbstractThe use of a biomarker is mandatory for quantitative analysis of exposure to secondhand smoke (SHS). This article summarizes urinary biomarkers of smoke exposure which can be now quantified. The most reliable urinary biomarkers to assess the exposure to SHS are NNAL 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanol and NNAL‐Glucuronides, which is metabolites of tobacco‐specific nitrosamine. These substances were detected even in 50% of children who had undetectable level of cotinine (<0.5 ng/ml). Urinary cotinine, which is determined by a highly sensitive competing enzyme immunoassay, is also a useful biomarker. However, individual variability of CYP2A6 allele,in which nicotine is catalyzed to cotinine, affects the level of urinary cotinine. Approximately 20% of Japanese subjects have homozygotes or heterozygotes of the CYP2A6*4 allele, which has impaired nicotine metabolism and subsequently may underestimate the actual exposure to SHS. In assessing the exposure to SHS, therefore, individual variability of CYP2A6 gene polymorphism should be taken into consideration. The combination of urinary cotinine measurement and self‐report of parents' smoking seems to be accurate to assess the exposure to SHS in mass screening. J. Clin. Lab. Anal. 25:354–358, 2011. © 2011 Wiley‐Liss, Inc.