Diagnostic screening of the congenital disorders of glycosylation (CDG) generally involves isoelectric focusing of plasma transferrin, a robust method easily integrated in medical laboratories. Structural information is needed as the next step, as required for the challenging classification of Golgi glycosylation defects (CDG-II). Here, we present the use of high-resolution nano liquid chromatography-chip (C8)-quadrupole time of flight mass spectrometry (nanoLC-chip [C8]-QTOF MS) for protein-specific glycoprofiling of intact transferrin, which allows screening and direct diagnosis of a number of CDG-II defects. Transferrin was immunopurified from 10 μL of plasma and analyzed by nanoLC-chip-QTOF MS. Charge distribution raw data were deconvoluted by Mass Hunter software to reconstructed mass spectra. Plasma samples were processed from controls (n = 56), patients with known defects (n = 30), and patients with secondary (n = 6) or unsolved (n = 3) cause of abnormal glycosylation. This fast and robust method, established for CDG diagnostics, requires only 2 hours analysis time, including sample preparation and analysis. For CDG-I patients, the characteristic loss of complete N-glycans could be detected with high sensitivity. Known CDG-II defects (phosphoglucomutase 1 [PGM1-CDG], mannosyl (α-1,6-)-glycoprotein β-1,2-N-acetylglucosaminyltransferase [MGAT2-CDG], β-1,4-galactosyltransferase 1 [B4GALT1-CDG], CMP-sialic acid transporter [SLC35A1-CDG], UDP-galactose transporter [SLC35A2-CDG] and mannosyl-oligosaccharide 1,2-alpha-mannosidase [MAN1B1-CDG]) resulted in characteristic diagnostic profiles. Moreover, in the group of Golgi trafficking defects and unsolved CDG-II patients, distinct profiles were observed, which facilitate identification of the specific CDG subtype. The established QTOF method affords high sensitivity and resolution for the detection of complete glycan loss and structural assignment of truncated glycans in a single assay. The speed and robustness allow its clinical diagnostic application as a first step in the diagnostic procedure for CDG defects.