BACKGROUND: Clinical detection of EGFR-TKI resistance mechanism through tissue can be really challenging due to risks associated with the procedure. Thus, liquid biopsy, especially circulation tumor DNA (ctDNA) analysis, can be an adequate source for biomarker testing in targeted therapy. Our study was aimed at clinical validation of liquid biopsy next-generation sequencing (NGS) by comparison with tissue biopsy, and we also investigated clinical utility of ctDNA NGS on the prediction of TKI outcomes. METHODS: Using hybrid capture panel NGS, we compared the concordance, sensitivity, and specificity of ctDNA using 39 paired plasma and tissue biopsy, and investigated the association between ctDNA genomic alterations of 147 first-generation TKI-relapsed patients and their response to first- and third-generation TKIs. RESULTS: The concordance for ctDNA and tissue biopsy was 84.62% among all patients, and even higher among late stage patients (88.24%). Among 147 EGFR-TKI-relapsed patients, T790M was the most common reason for resistance (40.13%). Compared with T790M-positive patients, patients only detected with sensitizing mutations (sensi-mutations) had lower mutant allele frequency (MAF) of sensi-mutations (P = 0.031). TP53 mutation showed negative impact on TKI treatments. In survival analysis of third-generation TKI, we found a positive correlation between ratio of T790M sensi-mutation and PFS (P = 0.018); also, higher MAFs of both sensi-mutation and T790M were observed in the PR group than the SD + PD group. CONCLUSIONS: Both ratio of T790M sensi-mutations and MAFs of EGFR mutations were associated with third-generation TKI outcomes. Thus, incorporation of high-throughput NGS into clinical trials may be crucial to identifying the response to osimertinib, as it provides more comprehensive genomic information. KEY POINTS: High concordance of ctDNA and tissue biopsy was observed. NGS of ctDNA from 147 TKI-relapsed patients showed that both high ratio of T790M sensitizing mutation (sensi-mutation) and high MAFs of mutations were all associated with better third generation TKI treatment outcomes. The quantification of both MAFs and T790M sensi-mutation ratio should be taken into consideration in some clinical situations, and incorporation of high-throughput NGS into clinical trials may be crucial to identifying the response to osimertinib, as it provides more comprehensive genomic information.