Mass spectrometry has become a popular analytical tool because of its high sensitivity and specificity. The use of a chiral derivatization reagent for the MS detection seems to be efficient for the enantiomeric separation of racemates. However, the number of chiral reagents for the LC-MS/MS analysis is very limited. According to these observations, we are currently underway the development of novel labeling reagents for chiral molecules in MS/MS analysis. The derivatization reagent that is effective for enhancing not only the ESI-MS/MS sensitivity but also the reversed-phase LC resolution of carboxylic acid enantiomers should have a highly proton-affinitive moiety and an asymmetric structure near the reactive functional group. Furthermore, the resulting derivative has to provide a characteristic product ion suitable for the selected reaction monitoring (SRM). Based upon these considerations, a series of prolylamidepyridine (PCP2, PCP3 and PCP4) was synthesized as ideal labeling reagents for LC-MS enantioseparation of chiral carboxylic acids and evaluated in terms of separation efficiency and detection sensitivity. Among the synthesized reagents, (S)-pyrrolidine-2-(carboxylic acid N-(pyridine-2-yl)amide (PCP2) was a most efficient chiral derivatization reagent for the enantioseparation of carboxylic acid. The Rs values and the detection limits of the derivatives of non-steroidal anti-inflammatory drugs (NSAIDs), which are selected as the representative carboxylic acids, were in the range of 1.35-4.72 and 50-260 amol, respectively. The PCP2 was applied to the determination of carboxylic acids in human saliva. Several biological carboxylic acids, such as lactic acid (LA), alpha-hydroxybutylic acid, maric acid, succinic acid and alpha-ketoglutalic acid, were clearly identified in the saliva of healthy persons and diabetic patients. Furthermore, the ratio of D-LA in diabetic patients was higher than that in normal person. Judging from these results, PCP2 seems to be a useful chiral derivatization reagent for the determination not only for chiral carboxylic acids but also achiral one.