We developed a fluorescence in situ hybridization (FISH) method for the rapid detection of Proteus spp. in urine, using a novel peptide nucleic acid (PNA) probe. Testing on 137 urine samples from patients with urinary tract infections has shown specificity and sensitivity values of 98 % (95 % CI, 93.2-99.7) and 100 % (95 % CI, 80,8-100), respectively, when compared with CHROMagar Orientation medium. Results indicate that PNA-FISH is a reliable alternative to traditional culture methods and can reduce the diagnosis time to approximately 2 h.