F2-isoprostanes (F2-iPs) comprise four classes of isomers produced non-enzymatically by free radical attack on arachidonic acid, a component of the cell membrane. This paper describes a new method for the quantification of F2-isoprostanes in urine samples from thoroughly diagnosed Alzheimer’s disease (AD) patients. The sample pretreatment consisted of liquid extraction of 900 μl urine with diethyl ether, its subsequent evaporation, and finally, reconstitution in 50 μl water. Of this, 20 μl was injected into a HPLC system with a 15 mm×1 mm porous graphitic carbon column coupled to a triple quadrupole mass spectrometer running in negative electrospray ionization mode. The F2-isoprostanes were separated in 15 min using a linear solvent gradient comprising water, methanol, acetonitrile and ammonium hydroxide at a pH of 9.5. The average recovery obtained was approximately 75%. The limit of detection (3S/N) was calculated for iPF2α-III to be 0.7 pg injected on column, corresponding to 0.1 nM. The average level of iPF2α was 241±163 pg/mg creatinine in the urine samples from AD patients (average±standard deviation). The corresponding control values were 216±101 pg/mg creatinine, i.e. no statistically significant difference was noticed. No correlation pattern specific to Alzheimer’s disease was revealed by principal component analysis of the isoprostane peaks obtained either. The results from this study support earlier findings that levels of peripheral isoprostanes are not increased in patients with Alzheimer’s disease.