Protein kinase is regarded as a potential target for anticancer therapeutics due to its relation to many diseases, in which more than one kinase participates in the cell signaling cascades. We herein demonstrate a multiplexed quantitative matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) approach to simultaneously assess the activity and inhibition of multiple protein kinases. In this design, substrate peptides phosphorylated by kinases of interest are specifically harvested by metal-organic framework (MOF)-templated porous CeO2 and consequently transformed to the dephosphorylated forms due to the phosphatase-like activity of CeO2, resulting in a unique quantitative MS signal with an enhanced intensity. Based on the peak area ratios of dephosphorylated variants of the phosphorylated product to respective deuterated internal standard, the activity and inhibition of each kinase can be independently profiled. In addition to the accurate characterization of protein kinase A (PKA) activity and inhibition induced by H-89, the multiplexing capability of the MS-based method allowed quantitative evaluation of the activity of Abl and Src, the two tightly associated kinases in the occurrence of chronic myeloid leukemia (CML) in a multiplexed format, exhibiting excellent orthogonality for the dual signal readout channels. Moreover, the inactivation of both Abl and Src by the inhibitor imatinib, dasatinib, and ponatinib was simultaneously traced, giving a whole picture of the competition behavior between the kinases for binding therapeutic molecules. This approach holds great promise in global investigation of kinase signal pathways and high-throughput screening of effective protein kinase inhibitors.