Lung cancer is the leading cause of cancer death worldwide.EGFR tyrosine kinase inhibitors (TKIs)gefitinib, erlotinib and afatinib, are approved in first line for metastatic Non-Small Cell Lung Cancer (NSCLC) with sensitizing epidermal growth factor receptor (EGFR) mutations. Intrinsic or acquired resistance to EGFR often related to RAS or secondary EGFR mutations, is a relevant clinical issue in NSCLC management. Although Src TK has been involved in such resistance in preclinical models, clinical development of Src inhibitors has been so far limited and unsuccessful in NSCLC therapy. To better define the molecular targets of the Src TKIs saracatinib, dasatinib and bosutinib, we used a variety of techniques including kinase assays, molecular modelling analysis, and in vitro/in vivo studies on NSCLC cell lines with different EGFR/RAS mutational profile and EGFR sensitivity to EGFR TKIs. Kinase inhibition assays supported by docking analysis demonstrated that all the compounds are able to directly inhibit not only Src, but also EGFR TK variants. However, in cell lysates only saracatinib efficiently reduced EGFR activation, while dasatinib was the most effective agent in inhibiting Src TK. Consistently, in EGFR-activating mutant, erlotinib sensitive cells, saracatinib showed anti-proliferative effects due to possible EGFR inhibition. In EGFR wt/RAS mutant cells, poorly dependent on EGFR activation and thus erlotinib resistant, Src inhibition by dasatinib interfered with cell proliferation and signal transduction. Based on these assumptions, we tested the following combinations: in EGFR-addicted cells, saracatinib with anti-EGFR drugs. (erlotinib or cetuximab), and in RAS mutant, erlotinib resistant models, dasatanib with the MEK inhibitor selumetinib. These combinations were effective both in vitro and in nude mice, inhibiting tumor growth, prolonging mice survival and interfering with signal transduction. Importantly, the combination of saracatanib and cetuximab was effective also in condition of T790M dependent EGFR resistance. In conclusion, Src inhibitors may act with different mechanisms, in NSCLC cell lines, depending on EGFR/RAS mutational profile. Integration of anti-Src agents with EGFR or MEK inhibitors could represent an effective therapeutic option for different cohorts of NSCLC patients.