Significance Allosteric sites in protein kinases offer opportunities for developing more selective inhibitors, but these sites are challenging to target because they involve protein–protein interfaces. We designed a site-directed approach to screen for molecules that bind to an allosteric peptide docking site on the protein kinase PDK1. We discovered molecules that structurally mimic the natural peptide ligand and inhibit PDK1 in cells. We also found that combining allosteric and ATP-competitive inhibitors completely blocked the activation of oncogenic kinases downstream of PDK1. This approach could be adapted to target an analogous allosteric site found on many other kinases.