As a key regulator of the B-cell receptor signaling pathway, Bruton's tyrosine kinase (Btk) has emerged as an important therapeutic target for various malignancies and autoimmune disorders. However, data on the expression profiles of Btk are lacking. Here, we report the discovery of a new, selective Btk probe and of a sandwich-type ELISA quantification method to detect endogenous Btk in live cells. We achieved selective labeling of Btk in vivo and quantified Btk levels in seven types of human lymphoma cell lines. This quantification method provides a powerful tool to study Btk in live cells that may also be useful in clinical settings.