Profiling of leaf extracts from mutants of Arabidopsis with defects in lipid desaturation demonstrates the utility of collision-induced dissociation time-of-flight mass spectrometry (CID-TOF MS) for screening biological samples for fatty acid compositional alterations. CID-TOF MS uses the collision cell of a quadrupole time-of-flight mass spectrometer to simultaneously fragment all of the ions produced by an ionization source. Electrospray ionization CID-TOF MS in the negative mode can be used to analyze fatty acyl anions derived from complex lipids as well as free fatty acids. Although acyl anion yield is shown to be a function of the lipid class and the position on the glycerol backbone, acyl compositional profiles can be determined, and the TOF detector provides resolution of nominally isobaric acyl species in the profiles. Good precision is obtained when data are acquired for approximately 1 min per sample.