All lung cancer patients with mutant epidermal growth factor receptor (EGFR) or rearranged EML4-ALK eventually develop acquired resistance to treatment. Rebiopsy may give insight into the resistance mechanisms and direct further lines of treatment. Here, we evaluate the potential interest and limitations of rebiopsy. Patients with mutant EGFR or rearranged EML4-ALK non-small cell lung cancer (NSCLC) and acquired resistance to tyrosine kinase inhibitors were included in a retrospective study to determine the percentage of patients who underwent rebiopsy and whether rebiopsy would have been possible, or not, in the remaining patients. In a cohort of 84 patients from 6 institutions, a biopsy had been performed in 39 cases. Biopsy samples were sufficient for histopathological or cytological examination in 35 cases (89.7 %). Complete or partial response had been observed in 84.5 % of patients whose cancer further progressed and who underwent rebiopsy. A biopsy could have been considered in 30 of the 45 remaining patients. Those with brain (N = 9) and bone (N = 2) metastases and/or with contraindications (N = 6) were excluded (two patients had both brain metastases and a contraindication). The rebiopsy target was thoracic in 62 % of cases and on distant metastases in 38 % of cases. Patients with NSCLC and an activating mutation could undergo a rebiopsy in 72 % of cases. A response to treatment does not preclude the possibility of rebiopsy at the time of progression.