Gene-sequencing projects have uncovered hundreds of sequences that possibly code for enzymes that cleave glycosidic linkages; the next challenge is to rapidly discover the chemical function of these gene products without individually testing every possibility. We have developed a sensitive new one-pot, high throughput strategy to address this problem with electrospray ionization mass spectrometry (ESI-MS). To this end, two small libraries of mass-differentiated glycosides, one with alpha-linked substrates and one with beta-linked substrates, have been synthesized. To validate the approach, the compound libraries were incubated with five different known glycosidases and the mixtures were monitored by ESI-MS. Only the known substrate was cleaved and no reaction inhibition by other library members was evident. This combined ESI-MS/library method was applied for the rapid identification of bacteria by profiling with glycosidase activity of cell extracts.