ABSTRACT Early diagnosis of urinary tract infections (UTIs) is essential to avoid inadequate or unnecessary empirical antibiotic therapy. Microbiological confirmation takes 24 to 48 h. The use of screening methods, such as cytometry and automated microscopic analysis of urine sediment, allows the rapid prediction of negative samples. In addition, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a widely established technique in clinical microbiology laboratories used to identify microorganisms. We evaluated the ability of MALDI-TOF MS to identify microorganisms from direct urine samples and the predictive value of automated analyzers for the identification of microorganisms in urine by MALDI-TOF MS. A total of 451 urine samples from patients with suspected UTIs were first analyzed using the Sysmex UF-1000 i flow cytometer, an automatic sediment analyzer with microscopy (SediMax), culture, and then processed by MALDI-TOF MS with a simple triple-centrifuged procedure to obtain a pellet that was washed and centrifuged and finally applied directly to the MALDI-TOF MS plate. The organisms in 336 samples were correctly identified, mainly those with Gram-negative bacteria (86.10%). No microorganisms were misidentified, and no Candida spp. were correctly identified. Regarding the data from autoanalyzers, the best bacteriuria cutoffs were 1,000 and 200 U/μl for UF-1000 i and SediMax, respectively. It was concluded that the combination of a urine screening method and MALDI-TOF MS provided a reliable identification from urine samples, especially in those containing Gram-negative bacteria.