Cell surface and extracellular proteins are O-glycosylated, where the most abundant type of O-glycosylation in proteins is the GalNAc attachment to serine (Ser) or threonine (Thr) in the protein chain by an a-glycosidic linkage. Most eukaryotic nuclear and cytoplasmic proteins modified by a-linked O-GlcNAc to Ser or Thr exhibit reciprocal O-GlcNAc glycosylation and phosphorylation during the cell cycle, cell stimulation, and/or cell growth. Less-investigated types of O-glycosylation are O-fucosylation, O-mannosylation, and O-glucosylation, but they are functionally of high relevance for early stages of development and for vital physiological functions of proteins. Glycosaminoglycans are a-linked to proteoglycans via a xylose-containing tetrasaccharide, represented by linear chains of repetitive disaccharides modified by carboxylates and O- or/and N-linked sulfates. Analysis of O-glycosylation by mass spectrometry (MS) is a complex task due to the high structural diversity of glycan and protein factors. The parameters in structural analysis of O-glycans include determination of (i) O-glycosylation attachment sites in the protein sequence, (ii) the type of attached monosaccharide moiety, (iii) a core type in the case of GalNAc O-glycosylation, (iv) the type and size of the oligosaccharide portion, (v) carbohydrate branching patterns, (vi) the site of monosaccharide glycosidic linkages, (vii) the anomericity of glycosidic linkages, and (viii) covalent modifications of the sugar backbone chains by carbohydrate- and noncarbohydrate-type of substitutents. Classical and novel analytical strategies for identification and sequencing of O-glycans by MS are described. These include methods to analyze O-glycans after total or partial release from the parent protein by chemical or enzymatic approach or to analyze O-glycosylated peptides by mapping and sequencing from proteolytic mixtures. A recombination process of multiply charged glycopeptides with electrons by electron capture dissociation Fourier transform ion cyclotrone resonance (FTICR)-MS has been introduced and is instrumental for nonergodic polypeptide backbone cleavages without losses of labile glycan substituents. A method for O-glycoscreening under increased sensitivity and efficient sequencing as a combination of an on-line coupling of capillary electrophoresis separation, as well as an automated MS-tandem MS (MS/MS) switching under variable energy conditions collision-induced dissociation (CID) protocol, is beneficial for determination of O-acetylation and oversulfation (Bindila et al., 2004a; Zamfir et al., 2004a). O-glycomics by robotized chip-electrospray/ionization (ESI)-MS and MS/MS on the quadrupole time-of-flight (QTOF) and FTICR analyzers, accurate mass determination, and software for assignment of fragmentation spectra represent essentials for high-throughput (HTP) in serial screenings (Bindila et al., 2004b; Froesch et al., 2004; Vakhrushev et al., 2005). Dimerization of intact O-glycosylated proteins can be investigated by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF)-MS after blotting.