NSD2 catalyzes the mono- and di-methylation of the e-amine of lysine 36 from histone H3, utilizing the methyl donor S-adenosyl-L-methionine. Increased catalytic activity of NSD2, either by overexpression or point mutations, is associated with multiple human cancers. To date, potent, selective and cell-active NSD2 inhibitors have not been described. A platform was established for the discovery of selective NSD2 inhibitors from quantitative high-throughput screening in a 1536-well format using a nucleosome substrate. Active compounds from the primary screen were triaged by a panel of assays and tested against two clinically relevant NSD2 mutants, E1099K and T1150A. Five confirmed inhibitors were further evaluated by a radiolabeled validation assay, surface plasmon resonance studies, methyltransferase profiling, and histone methylation in U-2 OS cells. Several inhibitors bound the NSD2 catalytic SET domain and demonstrated activity in cells, which validates the use of this workflow for the discovery and development of selective NSD2 inhibitors.