Abstract Prostate specific membrane antigen (PSMA), being both a biomarker and a therapeutic target, is important in prostate cancer (PC) diagnosis and treatment. Imaging and pathology are two of the most widely used approaches for PSMA detection in the diagnosis of PC. However, these techniques lack sensitivity for accurate assessment of low levels of PSMA expression and lack the capabilities to properly characterize heterogeneous PSMA expression. When using anti-PSMA imaging ligands, radiotherapies or immunotherapies, these drawbacks may ultimately prevent proper diagnosis and response prediction and could deny anti-PSMA treatment to qualified patients that have low PSMA expression. Better, more sensitive detection methods are needed for improved PSMA-based diagnosis and prediction of response. Quanticell is a novel histopathology technology utilizing fluorescent nanoparticles that are compatible with standard immunostaining practices. Combined with a dedicated software, Quanticell provides increased sensitivity and quantification and a wide dynamic range for improved detection and characterization of a broad range of target expression. A pilot study was conducted to demonstrate this technology on PSMA-expressing cell lines and clinical samples, and to evaluate its utility for discovery research and clinical studies. An anti-PSMA Quanticell protocol was developed and optimized on several PSMA-expressing cell lines, focusing on the lowest expressing sample. A dual-staining protocol for PSMA and a membrane marker was further developed to allow for detection and analysis of membrane-specific PSMA. Staining of negative and positive controls demonstrate high specificity to PSMA with high signal-to-noise that is not affected with increased primary concentration. When compared to DAB (diaminobenzidine) staining, we observed increased sensitivity in a low PSMA-expressing cell line, VCAP, indicated by increased detection of percent PSMA-positive cells. Additionally, using Quanticell, we demonstrate increased dynamic range in high expressing PSMA clinical samples compared to DAB staining, which is subject to saturation. Citation Format: Hemi Dimant, Ankit Gandhi, Micheal Maker, Adam Markman, Jeanette Reinhardt, Stefan Collins, Jacob Hesterman, Joseph Krueger, Kenneth Bloom, Julie Bailis, Gloria Juan. Quanticell, a novel histopathology technology for visualization and quantitation of membrane PSMA demonstrated in cell lines and clinical tissue [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3192.