This review evaluates the specificity and mechanism of action of 28 commercially available protein kinase inhibitors. The study examines their effects on a wide range of protein kinases to determine their true specificity. It is found that many of these inhibitors, such as KT 5720, Rottlerin, and quercetin, inhibit multiple kinases, sometimes more potently than their intended targets, leading to potential errors in interpreting their biological roles. In contrast, compounds like Ro 318220, H89, HA1077, and Y 27632 show greater selectivity but still inhibit multiple kinases. LY 294002 inhibits casein kinase-2 with similar potency to phosphoinositide 3-kinase. The most selective inhibitors are KN62, PD 98059, U0126, and PD 184352, which block the MAPK cascade by preventing the activation of MKK1, not by inhibiting MKK1 activity directly. The study highlights the importance of testing inhibitors against a broad panel of kinases to ensure their specificity. It also emphasizes the need for guidelines to use these inhibitors in cell-based assays to avoid misinterpretation of results. The findings suggest that the specificity of protein kinase inhibitors cannot be determined solely by their effect on closely related kinases. The study provides recommendations for the proper use of these inhibitors in cell-based assays to ensure accurate results.This review evaluates the specificity and mechanism of action of 28 commercially available protein kinase inhibitors. The study examines their effects on a wide range of protein kinases to determine their true specificity. It is found that many of these inhibitors, such as KT 5720, Rottlerin, and quercetin, inhibit multiple kinases, sometimes more potently than their intended targets, leading to potential errors in interpreting their biological roles. In contrast, compounds like Ro 318220, H89, HA1077, and Y 27632 show greater selectivity but still inhibit multiple kinases. LY 294002 inhibits casein kinase-2 with similar potency to phosphoinositide 3-kinase. The most selective inhibitors are KN62, PD 98059, U0126, and PD 184352, which block the MAPK cascade by preventing the activation of MKK1, not by inhibiting MKK1 activity directly. The study highlights the importance of testing inhibitors against a broad panel of kinases to ensure their specificity. It also emphasizes the need for guidelines to use these inhibitors in cell-based assays to avoid misinterpretation of results. The findings suggest that the specificity of protein kinase inhibitors cannot be determined solely by their effect on closely related kinases. The study provides recommendations for the proper use of these inhibitors in cell-based assays to ensure accurate results.