Our understanding of cancer has grown considerably with recent advances in high-throughput genome and transcriptome sequencing, but techniques to comprehensively analyze protein activity are still in development. Methods to quantitatively measure the activation of signaling pathways within tumors at baseline and following therapeutic intervention will prove critical to the design of proper treatment regimens. Focusing on breast cancer, we present such a method to understand kinase signaling using multiplexed kinase inhibitor beads coupled with mass spectrometry (MIB/MS).