The human epidermal growth factor receptor (HER) family comprises tyrosine kinase receptors that play a critical role in breast and gastric cancers. Lapatinib, a dual tyrosine kinase inhibitor (TKI) that targets HER1 and HER2 by binding to the ATP-binding site of their intracellular domains. Currently, this inhibitor is indicated in combination with capecitabine in advanced HER2-positive breast cancer after progression following standard treatment (anthracyclines, taxanes and trastuzumab) and in postmenopausal patients in whom hormonal therapy is indicated. Previously, our group had shown that HER2 was expressed and active (phosphorylated form) in the CSC subpopulation of several triple-negative and HER 2-negative mammary cancer cell lines. To study the implications of Lapatinib therapy in other breast tumor subtypes and its effect on CSCs, we proposed to study the effect of HER inhibitor therapy in different human and murine tumor cell models. In this study, we observed decreased viability in different breast cancer models including human (HCC70, HS578T) and murine (4T1, LM38-LP) triple-negative, estrogen-positive (MCF7) and HER2-positive (BT-474) cell lines upon lapatinib treatment. Similar effects were also found in the human hepatocellular carcinoma (Hep G2, HUH7) and prostate cancer (PC3) cell lines. Moreover, in different triple-negative breast cancer lines, a significant decrease in oncospheres (culture enriched in CSC) formation and size was also observed. Finally, in order to elucidate the underlying mechanisms, we compared the effects of lapatinib with Trastuzumab (monoclonal antibody against HER2) and Cetuximab (monoclonal antibody against EGFR). Lapatinib significantly affected cell viability in several cell lines and Cetuximab showed similar effects. However, trastuzumab showed no effect. Our findings, along with previous results, highlight the potential repositioning of lapatinib beyond HER2-positive breast cancer treatment.