Quantification of pristanic acid, phytanic acid, and very long chain fatty acids (i.e., hexacosanoic, tetracosanoic, and docosanoic acids) in plasma is the primary method for investigateing a multitude of peroxisomal disorders (PDs). Typically based on GC-MS, existing methods are time-consuming and laborious. In this paper, we present a rapid and specific liquid chromatography tandem mass spectrometric method based on derivatization with 4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(2-aminoethylamino)-2,1,3-benzoxadiazole (DAABD-AE). Derivatization was undertaken to improve the poor mass spectrometric properties of these fatty acids. Analytes in plasma (20 mul) were hydrolyzed, extracted, and derivatized with DAABD-AE in approximately 2 h. Derivatives were separated on a reverse-phase column and detected by positive-ion electrospray ionization tandem mass spectrometry with a 5 min injection-to-injection time. Calibration plots were linear over ranges that cover physiological and pathological concentrations. Intraday (n = 12) and interday (n = 10) variations at low and high concentrations were less than 9.2%. Reference intervals in normal plasma (n = 250) were established for each compound and were in agreement with the literature. Using specimens from patients with established diagnosis (n = 20), various PDs were reliably detected. In conclusion, this method allows for the detection of at least nine PDs in a 5 min analytical run. Furthermore, this derivatization approach is potentially applicable to other disease markers carrying the carboxylic group.