Class I phosphoinositide 3-kinases (PI3Ks) are lipid kinases that regulate multiple biological functions such as cell growth, proliferation, migration, and survival. Class I PI3Ks consist of four kinases isoforms. Over the past years many studies have documented that each isoform of PI3K plays specific biological functions in different cell types. Accumulating evidence indicates that activation of PI3K signaling is deregulated in human disease, including cancer. A major pharmaceutical effort has gone into developing PI3K inhibitors that hit multiple or individual PI3K isoforms, which are currently used in early and late-phase clinical trials. In this chapter we describe an in vitro PI3K assay that may be helpful in verifying which tumor cells have increased PI3K activity and thus may be targeted with inhibitors of PI3K.