PI3Kα has been identified as an oncogene in human tumors. By use of rational drug design, a targeted covalent inhibitor 3 (CNX-1351) was created that potently and specifically inhibits PI3Kα. We demonstrate, using mass spectrometry and X-ray crystallography, that the selective inhibitor covalently modifies PI3Kα on cysteine 862 (C862), an amino acid unique to the α isoform, and that PI3Kβ, -γ, and -δ are not covalently modified. 3 is able to potently (EC(50) < 100 nM) and specifically inhibit signaling in PI3Kα-dependent cancer cell lines, and this leads to a potent antiproliferative effect (GI(50) < 100 nM). A covalent probe, 8 (CNX-1220), which selectively bonds to PI3Kα, was used to investigate the duration of occupancy of 3 with PI3Kα in vivo. This is the first report of a PI3Kα-selective inhibitor, and these data demonstrate the biological impact of selectively targeting PI3Kα.