Lapatinib is a tyrosine kinase inhibitor of HER2 which blocks downstream signaling pathways in HER2 positive breast cancer cell lines, tumor xenografts and HER2 positive breast cancer patients. However, pre-clinical and clinical studies have shown that HER2 positive patients can exhibit either innate or acquired lapatinib resistance. Previous work in our laboratory resulted in the generation of lapatinib-resistant SKBR3 cells which exhibit decreased phosphorylation of eEF2 compared to parental cells. The aims of this study were to develop additional models of lapatinib resistance, characterise the cell lines and identify and validate alterations which may contribute to lapatinib resistance. HCC1954-L cells are a novel cell line model of acquired lapatinib resistance, developed through low-dose continuous conditioning with lapatinib. Using array CGH technology, HCC1954-L cells were found to have increased amplification and expression of STARD3. The decreased phosphorylation of eEF2 in SKBR3-L cells was found to be regulated by Ser359 phosphorylation of eEF2k in an mTOR-independent manner, suggesting a role for alterations in eEF2k and eEF2 in acquired lapatinib resistance. SILAC-proteomic analysis with SKBR3-par and SKBR3-L cells identified alterations in a number of different proteins of which CDK1, GAPDH, HER2, SELENBP1, SET and EBP1 were validated. Lapatinib conditioning of HCC1419 cells revealed a novel potential mechanism of lapatinib action – the induction of a senescent-like phenotype, which was reversed when lapatinib was removed from the media, suggesting a possible mechanism of lapatinib resistance, which may be dependent on p53 and p16 expression. Alterations in the phosphorylation of AKT, ERK, p70S6k and eEF2 correlated with lapatinib sensitivity in a panel of ~HER2-amplifed breast cancer cell lines. In conclusion, our data suggests several novel mechanisms of lapatinib resistance which warrant further investigation in in vivo models of HER2 positive breast cancer and ultimately in patients.