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Identification and antibiogram of uropathogenic microorganisms from direct urine samples present a great clinical impact. Here, we present a combined procedure to determine identification (IDd) of bacteria through MALDI-TOF-MS technology and antibiogram (ATBd) using disk-plate diffusion technique, of UTI-producing Enterobacterales against the most used antibiotics. Ninety-four urine samples with presence of pyuria and Gram-negative bacilli were selected. The IDd showed a high success rate (90%). ATBd procedure showed a high correlation for tested antibiotics. This simplified, low cost and reduced work time two-step procedure significantly reduces results turnaround time and benefit the clinical management of patients with UTI.