Background: The so-called “liquid biopsy” has become a powerful tool for cancer research in the recent years, with circulating cell-free DNA (cfDNA) that originates from tumors as one of the most promising analytes. In contrast to plasma derived cfDNA, only a few studies were performed investigating urinary cfDNA. One reason might be quick degradation and hence relatively low concentrations of urinary cfDNA. This study focused on examining the stability of cfDNA in urine using different ways of preservation under various storage conditions. Methodology: To mimic patient samples, a pool of healthy male and female urine donors was spiked with a synthetic cfDNA reference standard (fragment size 170 bp) containing the T790M mutation in the EGFR gene and preserved with three different buffers and no buffer over four different storage periods (0; 4; 12; 24 h) at room temperature vs. fridge. Preservatives used were the Urinary Analyte Stabilizer (UAS, Novosanis), the Urine Conditioning Buffer (UCB, Zymo) and a self-prepared buffer called “AlloU”. The cfDNA was extracted with the QIAamp MinElute ccfDNA Mini Kit (Qiagen) and the concentration measured with the Qubit™ 4 fluorometer (Thermo Fisher Scientific). Droplet digital PCR (ddPCR) was used for detection and quantification of the T790M mutation (Bio-Rad) Results: From the model samples with no preservation buffer, almost no spiked cfDNA could be recovered and the T790M variant could not be detected using ddPCR, indicating that without preservation cfDNA was degraded below the detection limit by the nucleases present in urine. The most effective stabilizing buffer over all storage periods and at room as well as fridge temperature was the UAS, resulting in accurate detection of the T790M variant using ddPCR. Conclusion: From a technical point of view, urinary cfDNA might be useful for non-invasive disease monitoring when the samples are stabilized and stored adequately during clinical routine. Citation Format: Carolin Münch, Saikal Shamkeeva, Mitja Heinemann, Berend Isermann, Sabine Kasimir-Bauer, Bahriye Aktas, Ivonne Nel. Stability of urinary cell-free DNA and detection of T790M variant. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6693.