A new and in part automated headspace solid-phase microextraction method for quantitative determination of the highly toxic rodenticide fluoroacetic acid (FAA) in serum and other biological samples has been developed. FAA and deuterated acetic acid (internal standard) were extracted from acidified samples by a StableFlex divinylbenzene–Carboxen on polydimethylsiloxane fibre. The acids were derivatised on the fibre in-situ with 1-pyrenyldiazomethane and detected using gas chromatography–mass spectrometry with electron impact ionisation and selected ion monitoring. The calibration curve for FAA in serum was linear over the range from 0.02 to 5 μg/ml, with limits of detection and quantification of 0.02 and 0.07 μg/ml, respectively. The method was also tested with spiked whole blood, urine, stomach contents and kidney samples. It was sufficiently reliable, reproducible and sensitive for use in routine forensic toxicology applications.