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With advances in targeted and personalized treatment for lung cancer, molecular analysis of tumors is routinely performed for sequencing of treatment options in patients with advanced non-small-cell lung cancer (NSCLC). Oncogene addiction due to driver mutations includes EGFR exon 20 insertion mutations, MET amplification, EML4-AL, KRAS G12C point mutations, RET rearrangements, HER2 amplification and mutations, and FGFR amplification and translocations. A re-biopsy at the time of tumor recurrence or progression after first-line treatment failure is important for further molecular assessment and personalized therapy. However, repeat tumor biopsies are fraught with challenges including access to the tumor, sample inadequacy, patient consent, patient performance status, safety, or physician’s choice or assessment. Cytological specimens are gaining importance but are limited due to validation difficulties. Liquid biopsies, which are minimally invasive have shown promise to assess dynamic biomarkers using ctDNA analysis and are thus frequently considered in routine clinical practice in advanced NSCLC patients to guide further targeted treatment. Here we present a comprehensive review that emphasizes the significance of performing tumor re-biopsy in advanced stage NSCLC patients following resistance to first-line treatment and simultaneously highlights the current challenges in performing the same and the current status and future perspectives of liquid biopsy in NSCLC.