B-cell precursor acute lymphoblastic leukemia (pre-B ALL) with mixed-lineage leukemia gene rearrangement (MLL-r) is a poor-prognosis subtype for which additional therapeutic targets are needed. To identify changes that could distinguish leukemic from normal precursor B-cells, we performed integrated multi-omics on primary patient samples. MLL-r cells feature an extensive remodelling of their glycoprotein glycocalyx, with increased Core 2 type O-glycans and complex N-glycans as well as significant changes in sialylation and fucosylation levels. Notably, a glycosaminoglycan remodelling from chondroitin sulfate to heparan sulfate was also observed. A survival screen to determine if glycan remodelling enzymes are redundant identified MGAT1 and NGLY1, components of the N-glycosylation/degradation pathway, as essential. OGT and OGA, unique enzymes that regulate intracellular O-GlcNAcylation, were also indispensable. Transcriptomics and proteomics further identified Fes and GALNT7-mediated glycosylation as possible therapeutic targets. Finally, next to well-known MLL-r pre-B ALL glycoprotein markers, our integrated multi-omics workflow identified previously unidentified diagnostic/therapeutic protein candidates.