A modified LC-MS/MS method for large-scale screening of metabolic phenotypes was developed and validated. Twenty amino acids and 5 metabolically related compounds are measured within 4 min using multiple reaction monitoring (MRM) transitions selective for each compound. Separation with a short C18 column and rapid gradient using the ion-pairing reagent perfluoroheptanoic acid allows chromatographic resolution of the isomers Ile and Leu and improved chromatographic peak shapes for Lys, Arg, and His. MRM transitions were established with capability to distinguish isomers Leu from Ile and Thr from homoserine even when chromatographic resolution is incomplete. The reproducibility of the assay was tested by adding eight stable isotope-labeled amino acid standards (AA*) to extracts of Arabidopsis thaliana seeds. In intra- and interday assay comparisons, mean coefficients of variation of the peak area ratios of AA/AA* were less than 5% for all but Gly/Gly-D2-15N1. Recoveries of these eight amino acids ranged from 62 to 94% and suppression of signal by the matrix were 31-65%. Dilution of seed extracts reduced ion suppression for early-eluting amino acids but had minimal effects for those eluting later. The intra- and interday accuracies for these eight amino acids were 77-131 and 88-133% of nominal concentrations, respectively.