Appropriate glycoprotein O -glycosylation is essential for normal development and tissue function in multicellular organisms. To comprehensively assess the developmental and functional impact of altered O -glycosylation, we have extensively analyzed the non-glycosaminoglycan, O -linked glycans expressed in Drosophila embryos. Through multidimensional mass spectrometric analysis of glycans released from glycoproteins by β-elimination, we detected novel as well as previously reported O -glycans that exhibit developmentally modulated expression. The core 1 mucin-type disaccharide (Galβ1-3GalNAc) is the predominant glycan in the total profile. HexNAcitol, hexitol, xylosylated hexitol, and branching extension of core 1 with HexNAc (to generate core 2 glycans) were also evident following release and reduction. After Galβ1-3GalNAc, the next most prevalent glycans were a mixture of novel, isobaric, linear, and branched forms of a glucuronyl core 1 disaccharide. Other less prevalent structures were also extended with HexA, including an O -fucose glycan. Although the expected disaccharide product of the Fringe glycosyltransferase, (GlcNAcβ1-3)fucitol, was not detectable in whole embryos, mass spectrometry fragmentation and exoglycosidase sensitivity defined a novel glucuronyl trisaccharide as GlcNAcβ1-3(GlcAβ1-4)fucitol. Consistent with the spatial distribution of the Fringe function, the GlcA-extended form of the Fringe product was enriched in the dorsal portion of the wing imaginal disc. Furthermore, loss of Fringe activity reduced the prevalence of the O -Fuc trisaccharide. Therefore, O -Fuc glycans necessary for the modulation of important signaling events in Drosophila are, as in vertebrates, substrates for extension beyond the addition of a single HexNAc.