© 2018, Refik Saydam National Public Health Agency (RSNPHA).Objective: Rapid identification of bacterial pathogens from urine specimens are essential to establish an adequate antibiotic therapy to treat urinary tract infections and it is very important in terms of cost.Urine culture is gold standard in the diagnosis of UTI (Urinary Track Infection).Urine culture and antibiogram usually takes 48-72 h.The bacteria produced in culture identified within a few minutes while the clinical samples are identified in a short time like two hours by Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS).The aim of this study is to contribute identification of the direct bacteria by MALDI-TOF MS system and to start early effective treatment of urine specimens. Methods: We analyzed 152 urine samples with UTI complaints were submitted to the microbiology laboratory between July to November 2015.While some of the samples were experimentally Gram stained, some were not.Formal acid extraction method was applied to urine specimens for direct bacterial identification with MALDI-TOF MS system.In this example, the urine culture was also performed simultaneously. Identification of the colonies in plaque,which was incubated in media at 37 °C in aerobic and 5% CO2 for 18-24 hours of incubation,were performed with the MALDI-TOF MS system. Results: The sensitivity of direct sample from MALDI-TOF MS system was found to be 76%.Statistical analysis was performed to determine whether the Gram stain had any effect on the bacterial load and consequently on the identification of the MALDI-TOF MS system.According to this, there was a significant difference between Gram staining group and non - Gram staining group (p < 0.001).Higher identification rates were found in Enterobactericeae, Enterocuccus faecalis, Staphylococcus aureus and Clostridium striatum (except streptococci and nonfermenter Gram negative bacteria), 105 cfu/mL single culture, MALDI-TOF MS directly from urine culture and MALDITOF MS from urine culture. Experimentally, in order to determine the safe identification by MALDI-TOF MS low bacterial concentrations; The E. coli and E. faecalis strains were identified by MALDI-TOF MS with1×106, 5×105, 2.5×105, 1.2×105,6×104 and 3×104 cfu/mL dilutions. A minimum of 1×106, 5×105 and 2×105 cfu/mL were identified to reliable scores for E. coli strains, while E. faecalis was defined at 1×106 cfu/mL dilution only. Conclusion: In this study, MALDI-TOF MS method the sensitivity 105 and / or 106 cfu/mL higher in the single kind of sample the Gram-mixed or 103 cfu/mL or less from the sample with bacteriuria and yeast identification was not possible. It was thought that Gram stain application determined the bacterial load before identification of direct urine specimen with MALDI-TOF MS system and contributed to the diagnostic performance of MALDI-TOF MS system and Gram stainings should be done for the samples taken for this reason. This method is faster in urine culture with 105 cfu/mL bacterial growth than conventional culture method.