This study describes a new method for simultaneous identification of uropathogens in the case of poly bacterial urinary tract infections. The method utilizes recently developed preparative isoelectric focusing (IEF) in cellulose-based separation medium with a subsequent analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Preparative IEF was successfully used for both purification and separation of bacteria, Escherichia coli (pI 4.6) and Staphylococcus aureus (pI 3.4), in urine samples. The focused zones of bacteria, localized by the positions of focused colored pI markers, were easily collected from the separation media after the IEF analysis and then unambiguously identified by MALDI-TOF MS. The proposed method enables the identification of bacteria in urine specimens when the concentration of individual bacteria is >= 10(4) cells mL(-1). Another benefit is the viability of bacteria extracted from the collected fractions after preparative IEF. (C) 2017 Elsevier B.V. All rights reserved.