Background: TNBC is a subtype of breast cancer negative for expression of estrogen and progesterone receptors and lack of HER2 gene amplification. It is associated with poor prognosis and lack of targeted therapies. Therefore, there is a need for identification of new therapies. EGFR, a member of the ErbB family, is frequently overexpressed in TNBC. Furthermore, crosstalk between EGFR and Src kinase signaling has been observed in breast cancer. The aim of this study is to assess the activity of afatinib, an irreversible pan-HER tyrosine kinase inhibitor alone and in combination with dasatinib, a Src inhibitor, in TNBC. Methods: Using proliferation assays, the effect of afatinib was assessed alone and in combination with dasatinib in TNBC cell lines. Combination indexes (CI) at ED₅₀, effective dose of combination that inhibits 50% of growth, were determined using the Chou and Talalay equation. CI < 1 implies synergy and CI > 1 implies antagonism. Results: The 6 TNBC cell lines tested responded to afatinib with IC₅₀ values ranging from 0.02 to 2.44 µM. As previously shown, the TNBC cell lines showed sensitivity to dasatinib with IC₅₀ values ranging from 9.6 nM to 3.1 µM. The HDQ-P1 cell line, classified as belonging to the basal-like 2 subtype of TNBC, showed significant sensitivity to both afatinib (IC₅₀ = 17.5 ± 2.1 nM) and dasatinib (IC₅₀= 16.6 ± 5.3 nM). The combination of afatinib and dasatinib was synergistic in 5 of the 6 TNBC cell lines tested (Table). Conclusions: Our results suggest that afatinib may have activity in TNBC. We also demonstrate that afatinib in combination with dasatinib may be more effective than either agent alone.