Protein phosphorylation carried out by protein kinases increases protein diversity and further influence various cellular physiology. Peptide substrates have been used for in vitro phosphorylation with purified kinase or cell lysates. Herein, a quantitative high-throughput platform was developed for assessing multikinase activity using nano-LC-MS/MS with a data-independent acquisition (DIA) approach. This platform was evaluated by studying the kinase activity in Taxol-treated SKOV3 cells. A library containing 38 peptide substrates was designed and analyzed to determine the activities of major kinases involved in cancer development. Our results showed that 23 synthetic peptide substrates had significance phosphorylated changes in triplicate biological experiments. The 23 peptide substrates were further verified by western blotting. Our findings reveal that Taxol suppressed SKOV3 cell survival by activating AMPK and suppressing the PI3K-Akt-dependent pathway, ultimately leading to mTOR inhibition. Furthermore, in combination with ERK, Akt, SGK, CK1 and ErbB2 inhibitors, Taxol enhanced the inhibitory effect on ovarian cancer. This platform can be an attractive approach for large-scale kinase activity studies to comprehensively elucidating the mechanisms of drug-disease treatments, enabling the investigation of more effective therapeutic strategies.