We describe a standardized approach for searching potent and selective inhibitors of glycosyltransferases by high throughput quantitative MALDI-TOFMS-based screening of focused compound libraries constructed by 1,3-dipolar cycloaddition of the desired azidosugar nucleotides with various alkynes. An aminooxy-functionalized reagent with a stable isotope was conjugated with oligosaccharides to afford glycopeptides as acceptor substrates with improved ion sensitivity. Enhanced ionization potency of new substrates allowed for MALDI-TOFMS-based facile and quantitative analysis of enzymatic glycosylation in the presence of glycosyl donor substrates. A non-natural synthetic sugar nucleotide was identified to be the first highly specific inhibitor for rat recombinant alpha2,3-(N)-sialyltransferase (alpha2,3ST, IC(50) = 8.2 microM), while this compound was proved to become a favorable substrate for rat recombinant alpha2,6-(N)-sialyltransferase (alpha2,6ST, K(m) = 125 microM). Versatility of this strategy was demonstrated by identification of two selective inhibitors for human recombinant alpha1,3-fucosyltransferase V (alpha1,3-FucT, K(i) = 293 nM) and alpha1,6-fucosyltransferase VIII (alpha1,6-FucT, K(i) = 13.8 microM).