Glycomics is an emerging research area. It aims to integrate large scale experimental data sets and computational techniques to better understand the structure, chemistry and roles of carbohydrates in guiding glycan formation. This in turn regulates diverse processes including cell adhesion, bacterial/viral interaction with host cells and cancer metastasis. The glycan structures are formed by catalytic action of glycosyltransferases enzymes on protein, lipid and small molecule scaffolds. The levels of these enzymes in the Golgi are a key feature regulating glycan structures. Abnormal glycosyltransferase activity is also associated with cancer. In the current paper, we present the development of a methodology to quantify glycosyltransferase activity in nanoliter scale reaction volumes in microarray format. Particular emphasis is placed on studies of enzymes mediating the O-linked glycosylation pathway in human cells. The method involves performing chemical/enzymatic reactions in small volumes for extended durations of time (30m-4h) with an array of carbohydrate substrates, followed by the rapid, tandem separation of product from unreacted radioactive sugar-nucleotide. Quantitative phosphorimaging provides estimation of reaction rate constants. Data are presented with cloned enzymes and with extracts from various cell systems consisting of human leukocytes, metastatic cell lines and stem cells. The activities of 4 families of enzymes, sialyltransferase, fucosyltransferase, galactosyltransferase and N-acetylglucosaminyltransferase, are monitored in these assays. The development of this method, will overcome complex and time-consuming synthesis and separation steps that typically limit the number of runs performed in traditional glycosyltransferase assays. This then allows the comparison of different enzyme families within a single cell system under uniform conditions using a diverse panel of substrates. The use of our novel assay to determine rate limiting steps in glycosylation reaction networks, and insight related to cellular development process will be discussed.