In lapatinib-resistant HER2-positive breast cancer cells, PI3K/AKT and MAPK signaling was maintained despite sustained suppression of the HER2 tyrosine kinase, and the tyrosine phosphorylated proteomes of both sensitive and drug-resistant cells were analyzed by immunoenrichment mass spectrometry, which revealed that the phosphorylation of Src family kinases and presumptive Src substrates in some resistant cell lines was increased. This evidence concerns the gene AKT1 and breast cancer.