Abstract We developed a headspace solid-phase microextraction (headspace SPME) method to measure acrolein in human urine. This new technique resolves some problems with the headspace gas chromatography and mass spectrometry (GC–MS) method which we developed previously. With the original method, a column and a filament were damaged by the injection of air. A 0.5-ml urine (or phosphate-buffered saline) sample in a glass vial containing propionaldehyde as an internal standard was heated for 5 min. The SPME fiber (65 μm carbonwax–divinylbenzene fiber) was exposed to the headspace and then inserted into a GC–MS instrument in which a DB-WAX capillary column (30 m×0.32 mm, film thickness 0.5 μm) was installed. The total analysis time was 15 min. The inter-assay and intra-assay coefficients of variation were 10.07 and 5.79%, respectively. The calibration curve demonstrated good linearity throughout concentrations ranging from 1 to 10 000 n M. The headspace SPME method exhibits high sensitivity and requires a short analysis time as well as the previous method. We conclude that this method is useful to measure urinary acrolein.