Amino acidopathies are a class of inborn errors of metabolism (IEM) that can be diagnosed by analysis of amino acids (AA) in plasma. Current strategies for AA analysis include cation exchange HPLC with post-column ninhydrin derivatization, GC-MS, and LC-MS/MS-related methods. Major drawbacks of the current methods are time-consuming procedures, derivative problems, problems with retention, and MS-sensitivity. The use of hydrophilic interaction liquid chromatography (HILIC) columns is an ideal separation mode for hydrophilic compounds like AA. Here we report a HILIC-method for analysis of 36 underivatized AA in plasma to detect defects in AA metabolism that overcomes the major drawbacks of other methods. A rapid, sensitive, and specific method was developed for the analysis of AA in plasma without derivatization using HILIC coupled with tandem mass-spectrometry (Xevo TQ, Waters). Excellent separation of 36 AA (24 quantitative/12 qualitative) in plasma was achieved on an Acquity BEH Amide column (2.1×100 mm, 1.7 μm) in a single MS run of 18 min. Plasma of patients with a known IEM in AA metabolism was analyzed and all patients were correctly identified. The reported method analyzes 36 AA in plasma within 18 min and provides baseline separation of isomeric AA such as leucine and isoleucine. No separation was obtained for isoleucine and allo-isoleucine. The method is applicable to study defects in AA metabolism in plasma.