Bacterial biofilms are responsible for the failure of many medical devices such as urinary catheters and are associated with many infectious and non-infectious complications. Preclinical and clinical evaluation of novel catheter coatings to prevent these infections needs to accurately quantify the bacterial load in the biofilm in vitro and ex vivo. There is currently no uniform gold standard for biofilm quantification for different surfaces and established biofilms. We have tried to establish a simple, accurate and reproducible method for extraction and measurement of biofilm bacteria on indwelling catheters, using a combination of vortexing and sonication. We demonstrate the usefulness of this method for catheters of different sizes - 3 Fr to 14 Fr - in vitro, in murine and porcine models, and indwelling in human clinical subjects. We also demonstrate consistent results with complex and polymicrobial biofilms. We believe that this standardized reproducible method will assist the assessment of biofilms in general and urological devices in particular in efforts to harness novel technologies to prevent healthcare associated infections.