Great effort has been devoted to characterize signaling lipids in central nervous system. This has led to a search for novel strategies to characterize hitherto unknown lipid compositions. Here we developed two methods, one for identification and one for quantification, for N-acyl amino acids, a novel lipid family. The identification method contains a series of purification steps followed by nano-LC/MS/MS and high-throughput screening of the datasets with a potent search algorithm based on fragment ion analysis. MS/MS spectra with good quality can be obtained with 150 fmol of targeted lipids on column with our nano-LC/MS/MS. More than one thousand mass spectra generated using the information dependent acquisition mode of Analyst QS software can be analyzed in 1 min using our home built software. The quantification method utilized the multiple reaction monitoring mode in Analyst software to measure the endogenous levels of N-acyl amino acids in rat brain. Using these two methods we were able to identify and quantify 11 previously reported N-acyl amino acids with endogenous levels ranging from 0.26 to 333 pmol g(-1) wet rat brain.