To identify novel tyrosine kinase substrates that have never been implicated in cancer, we studied the phosphoproteomic changes in the MCF10AT model of breast cancer progression using a combination of phosphotyrosyl affinity enrichment, iTRAQ technology, and LC-MS/MS. Using complementary MALDI- and ESI-based mass spectrometry, 57 unique proteins comprising tyrosine kinases, phosphatases, and other signaling proteins were detected to undergo differential phosphorylation during disease progression. Seven of these proteins (SPAG9, Toll-interacting protein (TOLLIP), WBP2, NSFL1C, SLC4A7, CYFIP1, and RPS2) were validated to be novel tyrosine kinase substrates. SPAG9, TOLLIP, WBP2, and NSFL1C were further proven to be authentic targets of epidermal growth factor signaling and Iressa (gefitinib). A closer examination revealed that the expression of SLC4A7, a bicarbonate transporter, was down-regulated in 64% of the 25 matched normal and tumor clinical samples. The expression of TOLLIP in clinical breast cancers was heterogeneous with 25% showing higher expression in tumor compared with normal tissues and 35% showing the reverse trend. Preliminary studies on SPAG9, on the other hand, did not show differential expression between normal and diseased states. This is the first time SLC4A7 and TOLLIP have been discovered as novel tyrosine kinase substrates that are also associated with human cancer development. Future molecular and functional studies will provide novel insights into the roles of TOLLIP and SLC4A7 in the molecular etiology of breast cancer.