Abstract Here we address some of the existing challenges in liquid biopsy workflows which can potentially benefit urine analysis from bladder cancer patients because approximately 1/4th of all human cancers are urological tumors. Globally, bladder cancer is the 10th most prevalent, and the 9th cause of mortality due to malignancy. The challenge addressed here is an improvement to sample preparation for sequencing. Among genders and disease states, urine samples vary in terms of cell count and hence, in DNA concentration. Therefore, it is critical to obtain consistent DNA fragments from variable samples across large cohorts of patients so that the sequencing leads to accurate identification of biomarkers of disease risk, progression, and treatment-response. In this direction, we present the advantages of a novel pixelated ultrasound technology which significantly improves sample-preparation in a very fast, economical, and high-throughput manner. Here, DNA is extracted from human urine samples and subjected to sonication to generate fragment sizes. Extracted DNA was loaded onto a regular 96-well round-bottom plate, which is sealed and placed inside the system where 12 columns of the 96-well plate are individually programmed and run simultaneously, hence enabling quick optimization. The system does not require time- and labor-intensive processing of the samples before or after the sonication which is required for other similar technology. Overall, the DNA samples were processed with significant improvement over other similar technology in terms of consistency, speed, and economy. Subsequently, the fragment sizes were analyzed. Analysis of results, as presented in the figures, show that the DNA fragment had the average size as predicted, uniform shearing, and very consistent across the 96-well plate containing different samples with no cross-contamination. The time, labour, expense, and risk turned out to be very low compared to other similar technology. The sizes were appropriate for library preparation and sequencing. The technology works consistently for both low and high cell numbers which is a critical factor for heterogenous samples from patients. In conclusion, the study presents improvements to library preparation for sequencing which fit into the larger workflow of non-invasive liquid biopsy using urine samples. The ability to obtain consistent DNA fragments in a quick and economical manner will greatly enable unique fingerprinting of an individual's cancer or cancer risk across large cohorts, with the potential for integration in automation workflows. Citation Format: Martha Evans-Holm, Nicolai Lumbres, Rwik Sen, Patricia Montilla-Perez, Marc A. Paradise, Peter Lentz. Improved high-throughput genomic analysis of urine samples using pixelated-ultrasound to enable personalized genomic profiling and therapeutic developments in 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 332.