This is the first report of an application of collisionally induced fragmentation of amino acids (AA) and their derivatives by MALDI TOF/TOF tandem mass spectrometry (MS). In this work, we collected the data on high-energy fragmentation reactions of a large group of protonated amino acids and their derivatives with the goal of determining which product ions are analyte specific and if yields of these fragment could be used for quantitative analysis. From 34 different amino acids (20 ␣-amino acids, -amino acids, homocysteine, GABA, and modified AA Met sulfone and sulfoxide, hydroxyproline, etc.) we observed that high yields of the target specific immonium ions and fragmentation patterns are most similar to EI or FAB CID on sector instruments. The major exceptions were two highly basic amino acids, Arg and Orn. It is noted that neither -, ␥-, nor ␦-amino acids produce immonium ions. As might be predicted from high-energy CID work on peptides from the sectors and TOF/TOF, the presence of specific indicator ions in MALDI tandem MS allows distinguishing isomeric and isobaric amino acids. These indicator ions, in combination with careful control of data acquisition, ensure quantitative analysis of amino acids. We believe our data provide strong ince their introduction in the late 1980s, the soft-ionization techniques, electrospray ionization (ESI) and matrix-assisted laser desorption/ ionization (MALDI), have became the major ionization methods for the analysis of biological molecules. While analysis of large biopolymers is successfully performed by both techniques, analysis of low molecular weight (LMW) molecules such as amino acids, pharmaceuticals, hormones, etc. is predominantly performed by ESI mass spectrometry with quadrupole mass analyzers. The major reasons not to use MALDI have been the abundance of matrix ions in the low mass region (100 to 600 u) and the lack of reasonable precursor selection in a MALDI tandem instrument. Also, until recently, MALDI was not considered to be a technique capable of providing robust quantitative data. Current developments in MALDI hardware [1,2] and increased attention paid to sample preparation have changed the situation. A number of recent publications describe application of MALDI TOF for quantitative studies [3][4][5][6][7][8], including favorable comparisons of MALDI TOF with the more traditional ESI LC/MS quantitation schemes [9 -11]. When installed on QqQ instruments, a MALDI source demonstrated good comparability with an ESI source on the same instrument in quantitative analysis of LMW compounds [12].Amino acid analysis (AAA) is one of the areas where ESI LC/MS/MS was successfully applied. ESI LC/ MS/MS has become a standard analytical tool in the analysis of low molecular weight physiological or therapeutic molecules of interest [13][14][15][16].We decided to examine if the combination of highenergy collision and prompt ion detection characteristic for MALDI TOF MS/MS could be applied for AAA. The proposed application of MALDI TOF for AAA contains some advantages o...