Urinary tract infections represent one of the most prevalent bacterial diseases, yet current diagnostic and research methodologies are hampered by inadequate culture media that fail to replicate the bladder biochemical environment. Conventional artificial urine formulations contain undefined components, lack essential nutrients, or inadequately support urinary microbiome (urobiome) growth. To address these limitations, we developed SimUrine, a fully defined synthetic urine medium that aims to replicate human bladder chemistry while supporting diverse microbial growth requirements. SimUrine was systematically developed through iterative optimization of multi-purpose artificial urine, incorporating defined concentrations of carbon sources, vitamins, trace elements, and amino acids within physiologically relevant ranges. The modular design enables component substitution without complete reformulation, facilitating customization for culturomics, antimicrobial susceptibility testing, and microbial ecology studies, while reducing batch-to-batch variability associated with authentic urine. Performance evaluation demonstrated SimUrine's capability to support the growth of fastidious urobiome members, including Lactobacillus species, Aerococcus urinae , and Corynebacterium riegelii , which fail to proliferate in conventional minimal media. Physicochemical characterization confirmed that SimUrine formulation exhibits properties within normal human urine ranges for density, conductivity, osmolarity, and viscosity, ensuring physiological relevance. Clinical applications revealed reduced antibiotic susceptibility compared to standard media, suggesting a more accurate representation of in vivo conditions. Co-culture experiments using Escherichia coli and Enterococcus faecalis demonstrated previously unobserved microbial interactions, highlighting SimUrine's utility for investigating urobiome dynamics. SimUrine represents a significant advancement in urobiome research methodology, providing a standardized, reproducible platform for investigating the urobiome under physiologically relevant conditions, potentially improving fundamental understanding and clinical diagnostic approaches. IMPORTANCE Urinary tract infections (UTIs) affect millions globally, yet current research and diagnostic methods rely on inadequate culture media that fail to replicate the bladder's unique biochemical environment. This fundamental limitation has hindered accurate UTI research and potentially compromised clinical treatment decisions. SimUrine addresses this critical gap as the first fully defined synthetic urine medium that mimics human bladder chemistry while supporting the growth of diverse urinary microbes. The breakthrough enables the cultivation of urobiome organisms in a minimal medium that resembles natural conditions, revealing novel microbial interactions that influence urinary health. Crucially, SimUrine demonstrates different antimicrobial susceptibility patterns compared to standard clinical media, suggesting current testing protocols may inaccurately predict treatment outcomes. This standardized, reproducible platform eliminates the variability of authentic urine samples while maintaining physiological relevance, potentially transforming urobiome research methodology and providing a new tool for the study of UTIs worldwide.