Animal models for host-microbial interactions have proven valuable, yielding physiologically relevant data that may be otherwise difficult to obtain. Unfortunately, such models are lacking or nonexistent for many microbes. Here, we introduce organ agar, a straightforward method to enable the screening of large mutant libraries while avoiding physiological bottlenecks. We demonstrate that growth defects on organ agar were translatable to colonization deficiencies in a murine model. Specifically, we present a urinary tract infection agar model to interrogate an ordered library ofProteus mirabilistransposon mutants, with accurate prediction of bacterial genes critical for host colonization. Thus, we demonstrate the ability ofex vivoorgan agar to reproducein vivodeficiencies. This work provides a readily adoptable technique that is economical and uses substantially fewer animals. We anticipate this method will be useful for a wide variety of microorganisms, both pathogenic and commensal, in a diverse range of model host species.