Background: RP-3500 is a selective and potent oral ATRi in development for advanced solid tumors harboring loss-of-function (LOF) alterations in genes associated with ATRi sensitivity. We determined whether ctDNA can facilitate enrollment/monitoring of pts treated with RP-3500. Methods: Serial plasma samples collected at baseline (BL, 99 pts) and early timepoints on therapy (89 pts, 3-9 weeks [wks]) were profiled for ctDNA (Tempus xF or Guardant360). Targeted next generation sequencing (NGS) (SNiPDx panel) was performed on matched peripheral blood mononuclear cells and tumor samples collected at BL. Molecular ctDNA response (MR) was defined as ≥50% reduction in mean variant allele frequency (VAF) from BL to any timepoint ≤9 wks on-therapy. Clonal hematopoiesis (CH) or germline alterations were excluded from the analysis. Efficacy was assessed in pts treated with > 100 mg RP-3500/day with ≥1 post-BL response assessment. Endpoints included progression-free survival (PFS) and clinical benefit rate (CBR; CR/PR by RECIST1.1 or PSA/CA-125, or > 16 wks on treatment). Results: BL ctDNA was detected in 82% (81/99) of pts. Eligibility alterations were evaluable by the ctDNA panel in 61% (60/99) of pts, excluding structural/copy number variants and genes/exons not on the panel. Percent agreement between BL ctDNA and local eligibility NGS test was 93% (56/60). CH variants were identified in 26 pts (1-14 per pt); median VAF was 0.4% (0.1-12.4%). Two pts with pathogenic ataxia-telangiectasia mutated (ATM) alterations were determined to be from CH. MRs were observed in 44% (24/55) of pts with median time to MR of 3.3 wks and were across tumors harboring ATM (10/20), BRCA2 (7/10), BRCA1 (4/15), CDK12 (1/3), PALB2 (1/3) and RAD51C (1/1) pathogenic alterations. Four pts with BRCA1 mutant tumors had MRs, 2 of whom (breast cancer) had received prior PARPi and had confirmed BRCA1 reversion mutations and clinical benefit (CB). One pt with gATM pancreatic cancer with CB had > 90% reduction in KRAS mutant VAF at 3 wks. MR was associated with longer mPFS (29 vs 12 wks, p = 0.0002) and significantly higher CBR (17/22 (77%) vs. 8/28 (29%); p = 0.001) than those without MR. Pts with MRs not achieving CB (N = 5) included 4 with RP-3500 dose interruptions/reductions and 1 who discontinued early (10 wks) due to clinical progression but with decreased target lesions and stable disease. Conclusions: ctDNA testing is a reliable method to detect DNA damage repair LOF alterations but is limited to alterations and genes/exons covered by the ctDNA test. CH alterations are frequent, especially for ATM, thus matched normal analysis is preferred. Changes in ctDNA as early as 3 wks were associated with improved outcomes and may be useful for evaluating drug activity in heterogenous tumors outside of traditional efficacy endpoints. Clinical trial information: NCT04497116.