Bladder cancer is the most common cancer of the urinary tract. More than 90% of bladder cancers are urothelial carcinoma, which are divided into non-muscle-invasive and muscle-invasive forms. Non-muscle-invasive tumors frequently recur (50-70%) and can also progress to invasion form (10-15%). These patients are monitored by cystoscopy and may have multiple resections over many years. Improved monitoring method is needed, ideally via urine analysis, which could reduce the morbidity and costs associated with long follow up. Currently there are no molecular biomarkers which could diagnose or accurately predict disease progression. We aimed to develop a clinically applicable, specific and sensitive panel of urine microRNAs enabling detect bladder cancer and predict risk of progression to muscle-invasive form.Within the exploratory phase of study we have analyzed expression profiles of 1733 miRNAs in urine supernatant of 16 bladder cancer patients (6 muscle invasive, 5 high-grade muscle non-invasive, 5 low-grade muscle non-invasive), 17 controls, 10 RCC patients and 4 urinary tract infections (UTI) using Affymetrix miRNA microarrays. Diagnostic and prognostic potential of selected microRNAs was further validated on independent samples in training phase (50 bladder cancer patients, 15 controls) and validation phase (100 bladder cancer patients, 55 controls, 45 renal cancer patients) using specific TaqMan assays and qRT-PCR method.Global expression profiling identified set of 76 miRNAs able distinguish bladder cancer patients from healthy controls (P < 0,01), thereof 64 highly up-regulated and 12 down-regulated. Moreover 23 miRNAs were able distinguish invasive and non-invasive forms of UCUB (P < 0,01) and 18 miRNAs high-grade and low-grad non-invasive (p < 0,01). Set of 12 miRNAs with highest expression level and statistical significance was validated in training phase of study. Based on the results the panel of three miRNAs (miR-31, miR-93, miR-191) was profiled. In validation phase we confirmed diagnostic potential and ability of this urine miRNA-based panel to diagnose patients with bladder cancer with high sensitivity and specificity (AUC = 0,8794, sensitivity = 82%, specificity = 80%).Our data have shown that urinary microRNAs could serve as sensitive and specific biomarkers of urinary bladder cancer and could be useful tool to increase sensitivity of standard cytological examination and reduce costs associated with long-term follow-up of bladder cancer patients. This work was supported by Ministry of Health of the Czech Republic, grant nr. 15-33158A, 15-34553A, 15-31627A, 15-34678A, 16-31314A, 16-31765A and by grant of Czech Grant Agency nr. 16-18257S.. All rights reserved. Citation Format: Jaroslav Juracek, Tana Machackova, Marek Vecera, Kamila Souckova, Jiri Sana, Parwez Ahmad, Natalia Anna Gablo, Ondrej Slaby, Michal Stanik, Jan Dolezel. Panel of urinary cell-free microRNAs in detection of urinary bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5451. doi:10.1158/1538-7445.AM2017-5451