The determination of enantiomeric excess (ee) of amino acids was achieved by investigating the collision-induced dissociation spectra of protonated trimers that were formed by electrospray ionization of amino acids in the presence of one of the following chiral selectors: L- or D-N-tert-butoxycarbonylphenylalanine, L- or D-N-tert-butoxycarbonylproline, and L- or D-N-tert-butoxycarbonyl-O-benzylserine. The protonated trimers were dissociated to form protonated dimers, and the observed dissociation efficiency r (i.e., the intensity ratio of protonated dimers to protonated trimers) for an enantiomeric mixture was found to be related to its ee value by the following equation: r = a + b/(c + ee), where a, b, and c were constants. A linear calibration plot was obtained by plotting r versus 1/(c + ee), where c was calculated with the MATLAB software, or by plotting 1/(r - r0) versus 1/ee, where r0 was the r value for the racemic mixture. The latter "two-reciprocal" method was more convenient for application. Another practical method for ee determination was the "three-point" method, whereby the ee of an unknown sample with a measured r value could be derived from the equation ee = 100¿1/(rL - r0) - 1/(rD - r0)¿/¿2/(r - r0) - 1/(rL - r0) - 1/(rD - r0)¿, with rL and rD being the r values for the enantiomerically pure L- and D-forms of the sample, respectively. A calibration plot was not required. The ee determination was achieved with acceptable precision even for the worst case of acceptable chiral recognition with a particular chiral selector, suggesting that the ee determination of all 19 common amino acids could be achieved by the present method. The ee of a histidine sample was determined both by the two-reciprocal method, giving an error of 0.2% ee (1.1% relative error) and consuming only approximately 5.3 nmol of sample, and by the three-point method, giving an error of 0.4% ee and consuming only approximately 2.3 nmol of sample. In the latter case, it took 27 min for the mass spectrometric measurements of the three calibration standards and an additional 9 min for the unknown sample. The direct ee determination of more than one amino acid in a mixture was also demonstrated in the study.