The diagnosis and follow-up of bladder cancer are mainly based on cystoscopy, an invasive method which could be negative in case of flat malignancies such as carcinoma in situ. Other noninvasive diagnostic methods have not yet given satisfactory results. There is a need for a reliable yet noninvasive method for the detection of bladder cancer. Our aim was to investigate whether cell-free DNA quantified in urine (ucf-DNA) could be a useful marker for the diagnosis of bladder cancer. A standard urine test was performed in 150 naturally voided morning urine samples that were processed to obtain a quantitative evaluation of ucf-DNA. Leukocyturia and/or bacteriuria were found in 18 subjects, who were excluded from the study. Statistical analysis was performed on 45 bladder cancer patients and 87 healthy subjects. Ucf-DNA was extracted from urine samples by a spin column-based method and quantified using four different methods: GeneQuant Pro (Amersham Biosciences, Pittsburg, PA, USA), Quant-iT DNA high-sensitivity assay kit (Invitrogen, Carlsbad, CA, USA), Real-Time PCR (Applied Biosystems, Foster City, CA, USA), and NanoDrop 1000 (NanoDrop Technologies, Houston, TX, USA). Median free DNA quantification did not differ statistically between bladder cancer patients and healthy subjects. A receiver-operating characteristic (ROC) curve was developed to evaluate the diagnostic performance of ucf-DNA quantification for each method. The area under the ROC curve was 0.578 for GeneQuant Pro, 0.573 for the Quant-iT DNA high-sensitivity assay kit, 0.507 for Real-Time PCR, and 0.551 for NanoDrop 1000, which indicated that ucf-DNA quantification by these methods is not able to discriminate between the presence and absence of bladder cancer. No association was found between ucf-DNA quantification and tumor size or tumor focality. In conclusion, ucf-DNA isolated by a spin column-based method and quantified by GeneQuant Pro, Quant-iT DNA high-sensitivity assay kit, Real-Time PCR or NanoDrop 1000 does not seem to be a reliable marker for the diagnosis of bladder cancer.