Background: Multiple biomarker testing strategies exist as the treatments of aNSCLC rapidly evolve. While upfront NGS is considered optimal, this practice has not been universally adopted. Surprisingly, the impact of NGS vs non-NGS approaches on OS is unclear, with the largest study to date demonstrating lack of benefit. We aim to identify the genomic testing strategy associated with the most optimal OS in aNSCLC using data from a large, contemporary electronic health record (EHR) database. Methods: This retrospective study used Flatiron EHR data. Patients diagnosed with aNSCLC between 11 Apr 2019 and 31 Dec 2021 who received systemic therapy were identified and followed from the date of diagnosis to 31 May 2022 or death. Testing methods reported within the patient’s chart were extracted. Methods assessed included NGS (defined as any test type with ‘NGS’ as test name) and non-NGS (defined as PCR, FISH, IHC, and other sequencing methods not containing ‘NGS’). Upfront testing was defined as tests received on or prior to the date of initiation of first-line (1L) therapy. Kaplan-Meier analyses were used to estimate and compare real-world overall survival (rwOS) by testing strategies. Among those with actionable genetic mutations, we also explored rwOS by timing of targeted therapy (TT) initiation. Results: Of a total 13,139 patients, mean age was 69, 49% female, 64% white, 33% Medicare insured, and 88% treated in community setting only. Median follow up time was 9 months. The percentage of patients with upfront NGS increased from 29% in 2019 to 66% in 2022. Median OS (mOS) was longest in patients with upfront NGS compared to those with upfront non-NGS genomic tests (p < 0.05), those with NGS after 1L (p < 0.05), and those with no NGS test ever (p < 0.05) (see table). 19% of patients with upfront NGS vs. 15% of those with upfront non-NGS received TT in 1L; 23% of patients with NGS vs. 19% of those with upfront non-NGS received TT at any line of therapy. In patients with actionable genetic mutations, mOS decreased with later initiation of TT; mOS was not reached (NR) (34.6 – NR) in patients who initiated TT in 1L, 34.7 (29.1 – NR) months in those with 2L TT, and 24.3 (20.3 – 30.1) months in those who initiated TT in 3L. Conclusions: This is the first and largest study to demonstrate that upfront NGS testing leads to better OS when compared to upfront non-NGS genomic testing, later NGS, or no-NGS in aNSCLC. In line with these results, we also found an OS benefit associated with earlier TT initiation. These results highlight the significant impact of upfront NGS testing and underscore for the need for both education and wider accessibility of NGS testing.