Despite high expectations for lung tumoroids, they have not been applied in the clinic due to the difficulty of their long-term culture. Here, however, using AO (airway organoid) media developed by the Clevers laboratory, we succeeded in generating 3 lung tumoroid lines for long-term culture (>13 months) from 41 lung cancer cases (primary or metastatic). Use of nutlin-3a was key to selecting lung tumoroids that harbor mutant p53 in order to eliminate normal lung epithelial organoids. Next-generation sequencing (NGS) analysis indicated that each lung tumoroid carried BRAF^G469A, TPM3-ROS1 or EGFR^L858R/RB1^E737*, respectively. Targeted therapies using small molecule drugs (trametinib/erlotinib for BRAF^G469A, crizotinib/entrectinib for TPM3-ROS1 and ABT-263/YM-155 for EGFR^L858R/RB1^E737*) significantly suppressed the growth of each lung tumoroid line. AO media was superior to 3 different media developed by other laboratories. Our experience indicates that long-term lung tumoroid culture is feasible, allowing us to identify NGS-based therapeutic targets and determine the responsiveness to corresponding small molecule drugs.