Acrylamide, classified by the IARC as a probable human carcinogen (Group 2A), is present in cigarette mainstream smoke and also some high-temperature-processed foods, thus smokers and consumers of certain foods are at risk of acrylamide exposure. The objectives of this study were to analyze N-acetyl-S-(propionamide)-cysteine (NASPC), an acrylamide metabolite, in the urine of smokers and nonsmokers, and to investigate the association between acrylamide exposure and urinary NASPC levels in smokers and nonsmokers in order to validate NASPC as a biomarker for the assessment of acrylamide exposure. Urine samples from 63 male military officers were collected as well as background personal information and smoking habits using questionnaires. Acrylamide exposure from tobacco smoke was represented by self-reported daily cigarette consumption and urinary cotinine levels. NASPC and cotinine were analyzed using our newly developed liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) methods. Our results reveal a statistically significant linear relationship between urinary NASPC and cotinine levels for smokers (Spearman correlation coefficient r=0.402, P=0.028), but insignificantly so for nonsmokers. These results verify our suggestion that urinary NASPC could serve as a sensitive, specific, noninvasive, and easily accessible biomarker for low-dose acrylamide exposure as also exposure to carcinogens in tobacco smoke. Routine monitoring of urinary NASPC could be used to assess human exposures to acrylamide in the living environment and the workplace.