The highly toxic nerve agent sarin (o-isopropyl methyl-phosphonofluoridate, GB) has been used in several armed conflicts and terror attacks in recent decades. Due to its inherent high sensitivity, liquid chromatography-mass spectrometry (LC-MS/MS) has the potential to detect ultratrace levels of fluoride-regenerated G and V agents after appropriate chemical derivatization. A new method for the retrospective determination of exposure to sarin was developed. The method is based on sarin regeneration from blood using the fluoride-induced technique followed by derivatization with 2-[(dimethylamino)methyl]phenol (2-DMAMP) and LC-ESI-MS/MS (MRM) analysis. The validated method presents good linear response in the concentration range of 5-1000 pg/mL with a limit of quantitation (LOQ) of 5 pg/mL, 13.8% accuracy, 16.7% precision and a total recovery of 62% ± 9%. This new analytical approach has several advantages over existing GC/GC-MS-based methods in terms of sensitivity, specificity and simplicity, in addition to a short LC-MS cycle time of 12 min. The method was successfully applied in an in vivo experiment for retrospective determination of sarin in a rabbit exposed to 0.1 LD50 sarin (1.5 µg/kg, i.v.). GB-2-DMAMP was easily determined in samples drawn up to 11 days after exposure. The high S/N ratio (500) observed for the GB-2-DMAMP signal in the 11day sample poses the potential for an extended time frame of months for analysis with this new method for the retrospective detection of sarin exposure. To the best of our knowledge, this is the first report on LC-MS/MS trace analysis of regenerated GB from biological matrices.