This study proposes an algorithm for microbiological diagnosis of urinary tract infections based on screening by luminometry and Gram-stain, followed by identification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Positive urine samples detected with the luminometry screening Coral UTI Screen TM system underwent Gram staining and identification of the causative organism was performed by MALDI-TOF Microflex LT mass spectrometer (Bruker Daltonics, Germany). Subsequently, the results were compared with those of conventional culture identification using WIDER MIC/id system (Francisco Soria Melguizo SA, Spain). Considering the conventional approach as the gold standard, the proposed algorithm presented both a high specificity (98.1%) and a positive likelihood ratio of 37.42. The implementation of this algorithm would allow diagnosis of urinary tract infection in less than an hour in 92.4% of positive samples. This combination of techniques would be useful particularly for patients with severe UTI, pyelonephritis or urinary sepsis.