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Aims: To reduce reporting time and to determine the exact prevalence of extended-spectrum β-lactamase (ESBL) enzymes and type of ESBL genes among single and multi-β-lactamase producing uropathogens. Methodology and results: Positive urine samples (20.8%) for significant pus cells and GNB were processed directly by modified CLSI phenotypic confirmatory test (MCPCT) for detection of ESBL enzymes and GNB isolates by PCR for ESBL genes. In MCPCT, cefotaxime and ceftazidime discs with and without clavulanic acid were used, dispensing 10 μL of both boronic acid and EDTA. Out of 378 GNB isolates tested, 167 (44.2%) were found positive for ESBL production by the modified CLSI phenotypic confirmatory test. Moreover, these GNB strains were assessed using uniplex and multiplex PCR, which also confirmed the findings of the modified CLSI phenotypic confirmatory test. CTX-M was the predominant gene, followed by TEM, OXA and SHV. Conclusion, significance and impact of study: The significance of the present study is that the modified CLSI phenotypic confirmatory test can be used to detect ESBL producers directly from clinical samples such as urine, cerebro-spinal fluid, blood, pleural fluid and ascetic fluid from which generally single organism is isolated. This method will not only save time but also help clinicians to start the treatment early so that the lives of critically ill patients can be saved. Moreover, MCPCT can provide a more accurate prevalence of ESBL producers than the CPCT method. [ABSTRACT FROM AUTHOR]