Treatment with anaplastic lymphoma kinase (ALK) inhibitors significantly improves outcome for non-small-cell lung cancer (NSCLC) patients with ALK-rearranged tumors. However, clinical resistance typically develops over time and, in the majority of cases, resistance mechanisms are ALK-independent. We generated tumor cell cultures from multiple regions of an ALK-rearranged clinical tumor specimen and deployed functional drug screens to identify modulators of ALK-inhibitor response. This identified a role for PI3Kβ and EGFR inhibition in sensitizing the response regulating resistance to ALK inhibition. Inhibition of ALK elicited activation of EGFR, and subsequent MAPK and PI3K-AKT pathway reactivation. Sensitivity to ALK targeting was enhanced by inhibition or knockdown of PI3Kβ. In ALK-rearranged primary cultures, the combined inhibition of ALK and PI3Kβ prevented the EGFR-mediated ALK-inhibitor resistance, and selectively targeted the cancer cells. The combinatorial effect was seen also in the background of TP53 mutations and in epithelial-to-mesenchymal transformed cells. In conclusion, combinatorial ALK- and PI3Kβ-inhibitor treatment carries promise as a treatment for ALK-rearranged NSCLC.