© 2016, The International Urogynecological Association. Introduction and hypothesis: We describe the rationale and methods of a study designed to compare vaginal and urinary microbiomes in women with mixed urinary incontinence (MUI) and similarly aged, asymptomatic controls. Methods: This paper delineates the methodology of a supplementary microbiome study nested in an ongoing randomized controlled trial comparing a standardized perioperative behavioral/pelvic floor exercise intervention plus midurethral sling versus midurethral sling alone for MUI. Women in the parent study had at least “moderate bother” from urgency and stress urinary incontinence symptoms (SUI) on validated questionnaire and confirmed MUI on bladder diary. Controls had no incontinence symptoms. All participants underwent vaginal and urine collection for DNA analysis and conventional urine culture. Standardized protocols were designed, and a central lab received samples for subsequent polymerase chain reaction (PCR) amplification and sequencing of the bacterial16S ribosomal RNA (rRNA) gene. The composition of bacterial communities will be determined by dual amplicon sequencing of variable regions 1–3 and 4–6 from vaginal and urine specimens to compare the microbiome of patients with controls. Sample-size estimates determined that 126 MUI and 84 control participants were sufficient to detect a 20 % difference in predominant urinary genera, with 80 % power and 0.05 significance level. Results: Specimen collection commenced January 2015 and finished April 2016. DNA was extracted and stored for subsequent evaluation. Conclusions: Methods papers sharing information regarding development of genitourinary microbiome studies, particularly with control populations, are few. We describe the rigorous methodology developed for a novel urogenital microbiome study in women with MUI.