Rapid diagnosis is one of the best ways to improve patient management and prognosis as well as to combat the development of bacterial resistance. The aim of this study was to study parameters that impact the achievement of reliable identification using a combination of flow cytometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF-MS).The study was carried out in nine hospitals in Spain and included 1,050 urine samples with bacterial counts of 5x106 bacteria/ml. MALDI-ToF-MS-based identification was performed according to a previously described protocol. Valid identification by direct MALDI-ToF-MS was obtained in 72.8% of samples, in 80.3% of samples found to be positive by culture, 32.2% of contaminated samples, and 19.7% of negative samples. Among the positives samples with a valid identification the concordance at the species level was 97.2%. The parameters related to success of direct identification were: high bacterial count, the presence of Escherichia coli as a pathogen and rod-bacteria morphology provided by flow cytometry. The parameters related to failure were a high epithelial cell (EC) count, a high white blood cell (WBC) count and urine samples obtained from in-patients. In summary, this multicentre study confirms previously published data on the usefulness and accuracy of direct MALDI-ToF-MS-based identification of bacteria from urine samples. It seems important to evaluate not only the bacterial count, but also other parameters, such as EC and WBC counts, bacterial species and morphology, and the health care setting, to decide whether the sample is suitable for direct identification.