Here, we describe an amino acid analysis that is based on the use of hydrophilic interaction liquid chromatography coupled with isotope dilution mass spectrometry for the accurate quantification of underivatized amino acids from hydrolyzed peptide/protein. Twelve underivatized amino acids were separated and detected during an 88-min runtime. The absolute limits of detection and limits of quantification (on column) of the four amino acids (isoleucine, phenylalanine, proline, and valine) were in the range of 6-80 and 20-200 fmol, respectively. As little as 25 pmol of peptide or protein hydrolysates is sufficient for determining absolute content.