Urinary tract infection (UTI) is one of the most common infections, accounting for a substantial portion of outpatient hospital and clinic visits. Standard diagnosis of UTI by culture and sensitivity can take at least 48 h, and improper diagnosis can lead to an increase in antibiotic resistance following therapy. To address these shortcomings, rapid bioluminescence assays were developed and evaluated for the detection of UTI using intact, viable cells of Photobacterium mandapamensis USTCMS 1132 or previously lyophilized cells of Photobacterium leiognathi ATCC 33981™. Two platform technologies—tube bioluminescence extinction technology urine (TuBETUr) and cellphone-based UTI bioluminescence extinction technology (CUBET)—were developed and standardized using artificial urine to detect four commonly isolated UTI pathogens—namely, Escherichia coli, Proteus mirabilis, Staphylococcus aureus, and Candida albicans. Besides detection, these assays could also provide information regarding pathogen concentration/level, helping guide treatment decisions. These technologies were able to detect microbes associated with UTI at less than 105 CFU/mL, which is usually the lower cut-off limit for a positive UTI diagnosis. Among the 29 positive UTI samples yielding 105–106 CFU/mL pathogen concentrations, a total of 29 urine specimens were correctly detected by TuBETUr as UTI-positive based on an 1119 s detection window. Similarly, the rapid CUBET method was able to discriminate UTIs from normal samples with high confidence (p ≤ 0.0001), using single-pot conditions and cell phone-based monitoring. These technologies could potentially address the need for point-of-care UTI detection while reducing the possibility of antibiotic resistance associated with misdiagnosed cases of urinary tract infections, especially in low-resource environments.