Lung cancer remains a leading cause of cancer-related mortality worldwide. Despite advancements in current therapies, the development of drug resistance and the need for improved treatment outcomes necessitate the exploration of novel therapeutic approaches. This study aimed to investigate the synergistic anti-cancer effects of Melittin, a bee venom peptide, in combination with Erlotinib, an EGFR inhibitor, in non-small cell lung cancer (NSCLC). The study evaluated the combined effects of Melittin and Erlotinib on A549 NSCLC cells. Cell viability, proliferation, migration, and apoptosis were assessed using standard in vitro assays. Mechanistic studies investigated the impact of the combination treatment on key signaling pathways, including those involving JAK2 and JAK3. Molecular docking simulations were performed to predict the binding interactions between Melittin and these kinases. The combination of Melittin and Erlotinib significantly inhibited A549 cell proliferation and migration, with a marked reduction in cell viability and enhanced apoptosis compared to either agent alone. Mechanistically, Melittin demonstrated interactions with JAK2 and JAK3, key proteins involved in apoptotic signaling. Molecular docking simulations further supported these findings, predicting strong binding affinities between Melittin and both kinases. These findings demonstrate a synergistic anti-cancer effect of Melittin and Erlotinib in A549 NSCLC cells. The observed interactions with JAK2 and JAK3 suggest a potential mechanism for Melittin's activity. These results highlight the potential of Melittin as a promising adjuvant to Erlotinib for the treatment of NSCLC.