To identify novel tyrosine kinase substrates that have never been implicated in cancer, we studied the phosphoproteomics changes in the MCF10AT model of breast cancer progression using a combination of phosphotyrosyl-affinity enrichment, iTRAQTM technology and LC-MS/MS. Using complementary MALDI- and ESI-based mass spectrometry, 57 unique proteins comprising of tyrosine kinases, phosphatases and other signaling proteins were detected to undergo differential phosphorylation during disease progression. Seven of these proteins (SPAG9, 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 EGF signaling and Iressa (Gefitinib). A closer examination revealed that the expression of SLC4A7, a bicarbonate transporter, was downregulated 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 to 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 would provide novel insights into the roles of TOLLIP and SLC4A7 in the molecular etiology of breast cancer.