The rapid development of molecular biology in recent years has allowed us to understand the main molecular steps involved in the development and progression of lung cancer. The identification of molecular alterations in specific tumor genes that function as key drivers for neoplastic growth has laid the foundations for new therapeutic approaches with targeted agents. An accurate detection of target mutation is mandatory for an efficient treatment. The main limitation of targeted therapies is the occurrence of acquired resistance that makes cancer unresponsive to treatment. In many cases, through the acquisition of additional (secondary) mutations the tumor is able to acquire the heterogeneity which may enable it to adapt to various conditions of the microenvironment, including those determined by the effect of treatment with specific drugs. New generation drugs are constantly under development to overcome tumor resistance and increase survival of lung cancer patients. In this process, a constant monitoring of the mutational status of the tumor is required. Different types of genetic alterations are involved in tumor development, progression, and induction of resistance, including single nucleotide variants, indels, amplifications, fusions etc. Mutation detection before first line treatment is usually performed on tissue or cytological samples. Resected tumor samples, biopsies and cytological specimens are available in about 25%, 35% and 40% of NSCLC patients, respectively. At progression, a re-biopsy should be obtained to detect the emergence of resistance-inducing mutations. Transbronchial tissue biopsy is the most common sampling method used for re-biopsy. However, several factors limit the success rate of re-biopsy, such as the performance status of the patient, the difficulty of accessing some tumor sites, and the invasiveness of sampling methods. When the amount and/or quality of the biological material is insufficient for molecular analysis, circulating free DNA (cfDNA) can represent a valid alternative in selected patients. Liquid biopsies have several advantages over tissue or cells: they are less invasive, can be repeated over time, and have a more rapid turnaround time. However, there are some critical issues that must be considered: 1) the possibility to detect a mutation in cfDNA is dependent on several clinicopathological parameters, including tumor type, tumor burden, and particularly tumor stage (a locally advanced tumor has a significantly lower probability to spread mutant DNA in the blood than a metastatic tumor); 2) a large amount of wild-type DNA circulates in the plasma with only trace amounts of the mutant allele; therefore, the analysis of genetic aberrations in cfDNA is challenging, requiring well standardized pre-analytical/analytical protocols and dedicated techniques with high sensitivity and specificity. Different technologies/protocols are required for the detection of these genetic aberrations. Robust and sensitive molecular biology techniques are nowadays available to detect mutations in driver genes before initiating a targeted treatment or to identify the emergence of secondary mutations at disease progression. The use of multi marker assays, and in particular next generation sequencing, is progressively becoming popular, allowing on one hand to reduce the working time, costs per single assay, and the amount of nucleic acids required for testing and increasing, in the other hand, throughput and overall quality. Recently, semi-quantitative or quantitative detection methods for the assessment of genetic aberrations in cfDNA have been developed with a number of potential clinical implications. An accurate quantification of mutated alleles in cfDNA during the first days of treatment could: a) complement or replace more expensive and invasive methods to assess response in treated patients; b) represent a new way to compare the effectiveness of different drug; c) be an additional tool to evaluate the best treatment regimen for patients. In addition, a periodic quantification of the mutation burden during all the treatment time could allow an early detection of resistance-inducing mutations for possible changes to therapy. Targeted Therapies, Acquired resistance, Lung cancer molecular biology