The PI3Ks (phosphatidylinositol 3-kinases) regulate cellular signalling networks that are involved in processes linked to the survival, growth, proliferation, metabolism and specialized differentiated functions of cells. The subversion of this network is common in cancer and has also been linked to disorders of inflammation. The elucidation of the physiological function of PI3K has come from pharmacological studies, which use the enzyme inhibitors Wortmannin and LY294002, and from PI3K genetic knockout models of the effects of loss of PI3K function. Several reports have shown that LY294002 is not exclusively selective for the PI3Ks, and could in fact act on other lipid kinases and additional apparently unrelated proteins. Since this inhibitor still remains a drug of choice in numerous PI3K studies (over 500 in the last year), it is important to establish the precise specificity of this compound. We report here the use of a chemical proteomic strategy in which an analogue of LY294002, PI828, was immobilized onto epoxy-activated Sepharose beads. This affinity material was then used as a bait to fish-out potential protein targets from cellular extracts. Proteins with high affinity for immobilized PI828 were separated by one-dimensional gel electrophoresis and identified by liquid chromatography–tandem MS. The present study reveals that LY294002 not only binds to class I PI3Ks and other PI3K-related kinases, but also to novel targets seemingly unrelated to the PI3K family.