BACKGROUND AND OBJECTIVE: Lung neoplasia is the leading cause of cancer death worldwide, thus, early detection and accuracy in establishing a diagnosis is paramount. As a consequence of decades of basic and translational studies revealing the genetic basis of lung cancer, a paradigm shift has occurred toward a personalized approach to medicine whereby mutational analysis confers an opportunity for safer, and expedient treatment options. In this context, next-generation sequencing (NGS) has emerged as a vital technological advance, and has become increasingly established as a core method for rapidly and effectively identifying actionable mutations in lung cancer. For these reasons, an updated review of the literature across invasive and non-invasive diagnostic modalities in lung cancer is warranted to inform diagnostic approaches and prompt new investigations. The objective of the present review is to provide a focused update on applications of NGS in lung cancer diagnostics, with a special focus on tissue acquisition methodologies and mutational analysis. METHODS: The search strategy included a survey of the current literature from 2005 to 2024 in PubMed, Medline, Scopus, and Google Scholar. Eligible study types included original research, literature reviews (narrative and systematic), and observational studies. which encompassed findings pertinent to the lung cancer diagnostics, mutational analysis and lung cancer treatment overlapping with applications and use of NGS technologies. KEY CONTENT AND FINDINGS: There are extensive and diverse advantages to the use of NGS in lung cancer diagnostics, especially when compared to traditional sequencing techniques including, speed, effectiveness, easy adoption in the context of analysis of samples prepared for lung cancer diagnosis. Advances in cell-free DNA reinforce the firm role of NGS in novel approaches. CONCLUSIONS: NGS implementation is a crucial and beneficial technological leap in lung cancer diagnosis, especially given the environment of novel and established targeted and immune based therapies which require mutational testing. Its numerous benefits such as expedient results and reduced sample requirements will continue to ability optimize lung cancer outcomes by virtue of improved patient safety, reduction of unnecessary procedures, and provision of accurate results.