Bruton's tyrosine kinase (BTK) is an important target for the treatment of multiple B-cell malignancies and autoimmune diseases. Development of approach that can accurately detect the expression and activity of BTK in live cells is of great significance for the diagnosis and treatment of cancer. In this study, we exploited ibrutinib, an FDA approved covalent targeting drug, for specific lanthanide labeling and quantification of BTK in live Ramos Burkitt’s lymphoma cells. A Europium (Eu) labeled ibrutinib probe (Ibt-DOTA-Eu) probe was first synthesized by conjugation of ibrutinib with DOTA, a chelator which can form highly stable complexes with lanthanides. We found that Ibt-DOTA-Eu is difficult to penetrate the cell membrane for in situ BTK labeling. To overcome this problem, we designed and synthesized an azide-modified ibrutinib probe (Ibt-N3) that can easily pass through the cell membrane, label BTK in live cells, and then realize DBCO-DOTA-Eu labeling through strain-promoted azide-alkyne cycloaddition (SPAAC) reaction. The Eu-labeled BTK in Ramos cells is quantified to be 61.28 ng/106 cells by using 153Eu-species-nonspecific-isotope-dilution ICP-MS coupled with HPLC. To our knowledge, this is the first study quantifying a disease-relevant kinase in live cells by using covalent targeting drug mediated lanthanide labeling and ICP-MS, which will prompt the application of ICP-MS in disease diagnosis and drug development in the future.