A structure-based design strategy was used to transform a promiscuous oxindole kinase inhibitor scaffold into the first cysteine-reactive, irreversible inhibitors of the human centrosomal kinase Nek2. These compounds achieve selective irreversible inhibition of Nek2 through alkylation of a key active-site cysteine Cys22). This cysteine is found in the glycine-rich loop of a small subset 2%) of human kinases. Many of the irreversible Nek2 inhibitors that we developed turned out to also be potent nM to pM affinity) inhibitors of the mitotic regulator Cdk1. Consistent with inhibition of Cdk1, these compounds rapidly triggered mitotic exit without cell division when added to cells arrested in mitosis. The induction of this dominant Cdk1-mediated phenotype limits the usefulness of these compounds with respect to studying the mitotic function of Nek2. However, our medicinal chemistry efforts led to the discovery of the propynamide oxindole compound 2 JH295). Compound 2 contains a key ethyl group that destabilizes binding to Cdk1, resulting in 2,000-fold loss of potency, while retaining nanomolar potency toward Nek2. At concentrations that give full inhibition of Nek2 in cells, compound 2 does not prevent mitotic progression or cause major defects in spindle assembly. These results suggest that compound 2 does not inhibit the mitotic regulatory kinases Cdk1, Aurora B, or Plk1 in cells. The development of several chemical and biological tools for the study of the cellular roles of Nek2 is also presented. These include new leads into Nek2 inhibitor scaffolds, an in vitro kinase assay to measure the binding affinity and alkylation efficiency of irreversible inhibitors, Nek2 active site-directed probes, cell-based Nek2 inhibition assays, cell lines expressing an inhibitor-resistant Nek2 mutant, and control compounds designed to tease out nonspecific effects of the active Nek2 inhibitors in cells.