Abstract Only a subset of patients with non-small cell lung cancer (NSCLC) respond to PD-L1 targeted therapy, even after solid tumor biopsy screening for PD-L1 expression. Downregulation of HLA I may contribute to this high prevalence of resistance, warranting its inclusion in attempts to increase response rates. While it is unclear if loss of HLA I occurs as a de novo or acquired resistance mechanism to immunotherapy, liquid biopsies could detect dynamic changes in expression levels, enable early detection of resistance, and accelerate the shift to alternate, more effective treatments. To quantify the expression of both PD-L1 and HLA I on circulating tumor cells (CTCs), we have developed automated exclusion-based sample preparation technology (ESP), which leverages the forces of surface tension and magnetism at the microscale for high-efficiency, high-throughput cell capture and manipulation. On-chip fluorescent antibody staining is performed for CTC identification, enumeration, and analysis of protein expression. A quantitative readout of protein expression is performed using a refined fluorescence microscopy approach wherein images of cells are acquired and analyzed with automated algorithms for uniformity and objectivity. To evaluate precision, accuracy, and analytical sensitivity of both ESP processing and quantitative microscopy, we used standardized cell lines, synthetic beads, and patient sample replicates. Calibration beads demonstrate high precision between multiple users for the quantitative microscopy (0.9% CV max of all wavelengths between 3 different analysts). ELISA beads with serial dilutions of protein standards demonstrate the accuracy of the quantitative microscopy (R2 values of 0.99 for both proteins). Cell lines demonstrate the precision of ESP for protein staining (3.4% CV max of two different cell lines tested on three different days). Patient sample replicates demonstrate precision for patients with as few as 3 CTCs per sample (6% CV max between triplicates). Early clinical utility of this assay was demonstrated on a preliminary patient cohort of 21 patient time points from 8 unique patients with NSCLC treated with immunotherapies. Phenotypic analysis for CTC expression of PD-L1 and HLA I with this assay demonstrated a sensitivity of 94% and specificity of 75% for detecting either resistant or stable disease during treatment with immunotherapies. Preliminary evaluation of patients with clear cell renal cell carcinoma demonstrates the feasibility of applying this approach to other cancer types. By confirming high performance, we ensure compatibility for clinical laboratory implementation and eventual application to better predict and detect resistance to PD-L1 targeted therapy in patients with NSCLC. The preliminary patient cohort suggests this diagnostic approach can detect fluctuations in expression of PD-L1 and HLA I in CTCs that also correspond to patient response to immunotherapy and warrants its pursuance in larger cohorts of patients with NSCLC and other tumor types. This abstract is also being presented as Poster B38. Citation Format: Jennifer L. Schehr, Zachery D. Schultz, Camila I. Hernandez, Matthew C. Mannino, Jay W. Warrick, Ticiana A. Leal, David J. Beebe, Joshua M. Lang. Analytical validation and preliminary clinical utility of PD-L1 and HLA I expression profiling of circulating tumor cells using automated exclusion-based sample preparation technology [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr PR10.