Background <p>Liquid biopsy approaches, especially the detection of circulating tumor DNA (ctDNA), are emerging as sensitive and reliable surrogates for tumor tissue-based routine diagnostic testing. Here, we retrospectively analyzed serially collected plasma samples of non-small cell lung cancer (NSCLC) patients obtained at first diagnosis to evaluate the added value of ctDNA analysis for detecting therapeutically relevant variants and determining the consequent clinical implications.</p> Methods <p>One hundred eighty plasma samples from consecutively recruited NSCLC patients were included. Circulating cell-free DNA (ccfDNA) was extracted and analyzed with the UltraSEEK Lung Panel v2 on the MassARRAY System. Tumor tissue next-generation sequencing (NGS) data, performed as routine molecular testing in the clinical setting, were retrieved from the national pathology registry for 132 patients.</p> Results <p>Here we show that in 82% of the patients, mutations are concordantly detected in tumor tissue and plasma. More mutations are reported with tumor tissue-based NGS in nineteen patients, while in four patients additional mutations are detected in plasma. Tissue-based molecular tumor profiling identifies 60 patients eligible for targeted treatment including fifteen (8%) harboring fusions currently not covered by UltraSEEK. Based on ctDNA analysis, 41 patients (23%) are identified as eligible for BRAF^V600-, EGFR-, or KRAS^G12C-targeted therapies. In the absence of tumor tissue NGS data (<i>n</i> = 48), five therapeutically relevant mutations are detected.</p> Conclusions <p>Molecular tumor profiling of ctDNA identifies therapeutically relevant mutations at a comparable rate to tumor tissue-based NGS and might therefore serve as an alternative or complementary test for the detection of actionable variants in plasma.</p>