<jats:title>Abstract</jats:title><jats:p>Mercapturic acids are highly important and specific biomarkers of exposure to carcinogenic substances in occupational and environmental medicine. We have developed and validated a reliable, specific and very sensitive method for the simultaneous determination of five mercapturic acids derived from several high‐production chemicals used in industry, namely ethylene oxide, propylene oxide, acrylamide, acrolein and <jats:italic>N,N</jats:italic>‐dimethylformamide. Analytes are enriched and cleaned up from urinary matrix by offline solid‐phase extraction. The mercapturic acids are subsequently separated by means of high‐performance liquid chromatography on a Luna C8 (2) column and specifically quantified by tandem mass spectrometric detection using isotopically labelled analytes as internal standards. The limits of detection (LODs) for <jats:italic>N</jats:italic>‐acetyl‐<jats:italic>S</jats:italic>‐2‐carbamoylethylcysteine (AAMA) and <jats:italic>N</jats:italic>‐acetyl‐<jats:italic>S</jats:italic>‐2‐hydroxyethylcysteine (HEMA) were 2.5 µg/L and 0.5 µg/L urine, while for <jats:italic>N</jats:italic>‐acetyl‐<jats:italic>S</jats:italic>‐3‐hydroxypropylcysteine (3‐HPMA), <jats:italic>N</jats:italic>‐acetyl‐<jats:italic>S</jats:italic>‐2‐hydroxypropylcysteine (2‐HPMA) and <jats:italic>N</jats:italic>‐acetyl‐<jats:italic>S</jats:italic>‐(<jats:italic>N</jats:italic>‐methylcarbamoyl)cysteine (AMCC) it was 5 µg/L. These LODs were sufficient to detect the background exposure of the general population. We applied the method on spot urine samples of 28 subjects of the general population with no known occupational exposure to these substances. Median levels for AAMA, HEMA, 3‐HPMA, 2‐HPMA and AMCC in non‐smokers (n = 14) were 52.6, 2.0, 155, 7.1 and 113.6 µg/L, respectively. In smokers (n = 14), median levels for AAMA, HEMA, 3‐HPMA, 2‐HPMA and AMCC were 243, 5.3, 1681, 41.7 and 822 µg/L, respectively. Due to the simultaneous quantification of these mercapturic acids, our method is well suited for the screening of workers with multiple chemical exposures as well as the determination of the background excretion of the general population. Copyright © 2008 John Wiley & Sons, Ltd.