Urinary tract infection is most frequently found bacterial infection in urinary tract of human beings and 75-95% of urinary tract infections are caused by uropathogenic . The uropathogenic bacteria produces several virulence factors, siderophore is one of them. Siderophore is iron acquisition protein and play significant role in pathogenicity of UTI’s by helping uropathogenic bacteria to persist in urinary tract of host cells in adverse conditions. The study was conducted at SRL laboratory in Ujjain Center from November 2018- October 2019. Midstream urine sample of suspected UTI patients were collected and was isolated and identified using standard microbiological procedure and after incubation, growth was observed. Colonies more than 10cfu/ml were considered as significant for UTI and detection of siderophore production in uropathogenic bacteria was done by using Chrome Azurol Assay (CAS). Out of 200 collected urine sample in 120 urine samples uropathogenic bacteria was identified and isolated. Among 120 isolates of siderophore production was observed in 48 isolates of , and their percentage was 40%. Iron acquisition protein or siderophores are a major virulence factor necessary for pathogenesis of UTI caused by and CAS assay is the most efficient method for detection of siderophore production in uropathogenic