The study examines the specificities of 28 commercially available compounds reported to be selective inhibitors of specific serine/threonine protein kinases. The compounds KT 5720, Rottlerin, and quercetin were found to inhibit many protein kinases, sometimes more potently than their presumed targets, leading to potentially erroneous conclusions in cell-based experiments. Ro 318220, bisindolylmaleimides, H89, HA1077, and Y 27632 were more selective but still inhibited two or more protein kinases. LY 294002 inhibited casein kinase-2 and phosphoinositide 3-kinase with similar potency. The most selective inhibitors were KN62, PD 98059, U0126, PD 184352, rapamycin, wortmannin, SB 203580, and SB 202190. U0126 and PD 184352 blocked the mitogen-activated protein kinase (MAPK) cascade by preventing MKK1 activation, not by inhibiting MKK1 activity directly. The results highlight that protein kinase inhibitor specificities cannot be assessed by studying their effects on closely related kinases. The authors propose guidelines for using protein kinase inhibitors in cell-based assays to avoid false conclusions.The study examines the specificities of 28 commercially available compounds reported to be selective inhibitors of specific serine/threonine protein kinases. The compounds KT 5720, Rottlerin, and quercetin were found to inhibit many protein kinases, sometimes more potently than their presumed targets, leading to potentially erroneous conclusions in cell-based experiments. Ro 318220, bisindolylmaleimides, H89, HA1077, and Y 27632 were more selective but still inhibited two or more protein kinases. LY 294002 inhibited casein kinase-2 and phosphoinositide 3-kinase with similar potency. The most selective inhibitors were KN62, PD 98059, U0126, PD 184352, rapamycin, wortmannin, SB 203580, and SB 202190. U0126 and PD 184352 blocked the mitogen-activated protein kinase (MAPK) cascade by preventing MKK1 activation, not by inhibiting MKK1 activity directly. The results highlight that protein kinase inhibitor specificities cannot be assessed by studying their effects on closely related kinases. The authors propose guidelines for using protein kinase inhibitors in cell-based assays to avoid false conclusions.