Abstract There is an unmet need for improved methods of rapidly identifying responses to targeted therapies. Liquid biopsy approaches have potential as early biomarkers of response based on noninvasive, real-time monitoring of disease burden. We have used the ultrasensitive targeted error correction sequencing (TEC-Seq) approach to analyze 58 cancer driver genes in patients with metastatic non-small cell lung cancer undergoing treatment with targeted tyrosine kinase inhibitors. As a proof-of-principle study, liquid biopsies were obtained from sixteen patients immediately prior to treatment, 6-22 days after treatment, and at serial timepoints until disease progression. Tumor derived alterations and copy number changes were directly detected in plasma and tracked over timepoints analyzed. Based on the dynamics of cell-free circulating tumor DNA (ctDNA) mutations identified, we developed a noninvasive measure of cell-free clonal tumor load (cfTL) to evaluate real-time response to treatment. These analyses revealed that patients with a radiographic response to therapy had a significant drop in cfTL from an average mutant allele fraction of 3.59% to 0.13% within 6-22 days (P < 0.05) as well as in the number of detectable mutations and aneuploidy scores, while radiographic non-responders had limited to no changes. Analyses of residual cfTL 6-22 days after treatment stratified patients into ctDNA responders and ctDNA non-responders. ctDNA responders had improved progression-free survival (12.4 vs 1.7 months, P < 0.001), which was detected on average 38 days earlier and was as predictive as CT imaging. These analyses provide an approach for rapid evaluation of response to targeted therapies and have important clinical implications for the management of cancer patients. Citation Format: Jillian A. Phallen, Alessandro Leal, Brian D. Woodward, Patrick M. Forde, Jarushka Naidoo, Kristen Marrone, Julie Brahmer, Jacob Fiksel, Doreen N. Palsgrove, Stephen Cristiano, Daniel Bruhm, Elizabeth Weihe, Vilmos Adleff, Parissa Keshavarzian, Valsamo Anagnostou, Robert B. Scharpf, Victor E. Velculescu, Hatim Husain. Early noninvasive prediction of response to targeted therapy in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4596.