Objectives: Re-biopsy in progressive advanced Non-Small-Cell Lung Cancer (NSCLC) after first line treatment may reveal information about evolving tumor biology during treatment. Our study aims to investigate the feasibility, risk of complications, and clinical relevance of performing re-biopsy systematically. Materials and methods: NSCLC patients with advanced, non-targetable disease, receiving first line systemic treatment, were included in a prospective single-centre study (NCT03512847). A diagnostic biopsy was performed at baseline and repeated at time of progression, preferentially from the progressive lesions as determined by CT or PET/CT. The primary endpoint was feasibility, including complication rate to re-biopsy. Secondary endpoints were clinical relevance, defined as a potential of changing treatment or follow-up, due to new histological evidence, specifically a change in PD-L1 Tumor Proportion Score (TPS). Results: Fifty-one patients with progressive advanced NSCLC had re-biopsy performed. Median time from patients’ acceptance to biopsy was seven days (range: 0–31). Complication rate was 6% (n = 3) represented by pneumothorax, hydro-pneumothorax and pneumonia, respectively. No severe or chronic complications occurred. Sufficient material for PD-L1 analyses was obtained in 46 of 51 patients: the remaining five cases had insufficient tissue for analyses, no malignant cells/only suspected malignant cells, questioning whether progression was real. PD-L1 TPS change was observed in 33% of patients (n = 15) and 17% (n = 8) had potentially clinically relevant changes. A significantly higher chance of PD-L1 TPS change was observed in chemotherapy-treated patients. Conclusion: Our study showed that re-biopsy is feasible, with low risk of complications, and can be clinically relevant in patients with suspected progression in advanced NSCLC.