Haspin/Gsg2 is a kinase that phosphorylates Histone H3 at Thr-3 (H3T3ph) during mitosis. Its depletion by RNA interference results in failure of chromosome alignment and a block in mitosis. Haspin therefore is a novel target for development of anti-mitotic agents. We report the development of a high throughput time-resolved fluorescence resonance energy transfer (TR-FRET) kinase assay for Haspin. Histone H3 peptide was used as a substrate, and a Europium-labeled H3T3ph phosphospecific monoclonal antibody was used to detect phosphorylation. A library of 137632 small molecules was screened at Km concentrations of ATP and peptide to allow identification of diverse inhibitor types. Reconfirmation of hits and IC50 determinations were carried out with the TR-FRET assay and by a radiometric assay using recombinant Histone H3 as the substrate. A preliminary assessment of specificity was made by testing inhibition of two unrelated kinases. EC50 values in cells were determined using a cell-based ELISA assay of H3T3ph. Five compounds were selected as leads based on potency and chemical structure considerations. These leads form the basis for the development of specific inhibitors of Haspin that will have clear utility in basic research and possible use as starting points for development of anti-mitotic anticancer therapeutics.