Abstract Non-small cell lung cancer (NSCLC) is the most frequent type of lung cancer, accounting for about 80 - 85% of all diagnoses. Unfortunately, 75% of NSCLC diagnoses are made when cancer has already spread and the prognosis is poor. Therefore, biomarker investigation that might allow for early detection of NSCLC is critical to improving patient outcomes. The purpose of this study is to demonstrate the feasibility of liquid biopsy to detect and identify circulating rare events, such as circulating tumor cells (CTCs), associated with NSCLC. Here we apply our non-enrichment-based High Definition Single Cell Assay (HDSCA) workflow which utilizes an immunofluorescence staining technique and an unsupervised rare event detection algorithm to identify the circulating rare cells and acellular events in samples collected from 206 NSCLC patients and 60 healthy individuals. We detected a heterogeneous population of circulating rare events in NSCLC, including epithelial, mesenchymal, and endothelial cells, as well as acellular events such as oncosomes. Cancer-associated circulating rare events with statistically significant prevalence in NSCLC compared to healthy individuals were observed. Molecular characterization using multiplexed targeted proteomics based on imaging mass cytometry and genomic analysis by single-cell copy number profiling confirmed the phenotypic heterogeneity and genomic profiles of the detected circulating rare events. In this study, our results demonstrate the use of the extended capabilities of liquid biopsy to identify a comprehensive overview of circulating rare events that can provide clinically valuable biomarkers in NSCLC. Citation Format: Jiyoun Seo, Stephanie N. Shishido, Jeremy Mason, Carmen Ruiz Velasco, Jaden Mullin, Rishvanth Prabakar, James Hicks, Peter Kuhn. Characterization of circulating rare events in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3698.