Sex differences in the gut microbiome may play an important role in sex-specific physiological processes and diseases. Yet, the underlying mechanisms that lead to sex differentiation of the gut microbiome remain poorly understood. Previous research demonstrated that microbial β-glucuronidase (GUS) enzymes metabolize glucuronidated estrogen, thus facilitating recirculation via the enterohepatic circulation process. In addition, studies have shown that there are differences in enzyme activity among microbial GUS enzymes from different bacteria suggesting the possibility of substrate specificity. However, there is a lack of studies exploring the bacterial metabolism of testosterone glucuronide, hindering our understanding of sexual differentiation in the gut microbiota. To bridge this gap, I investigated GUS enzyme substrate specificity using a coupled enzymatic assay. The coupled assay employs two distinct enzymes – microbial GUS enzyme to deconjugate estrogen and testosterone glucuronide, and uronate dehydrogenase enzyme (UDH) to generate end-point measurements. I optimized the assay by establishing conditions for the two uncoupled assays with GUS and UDH enzymes. GUS enzyme in recombinant form and E. coli displayed enzymatic activity in the 4-nitrophenol assay. Furthermore, I optimized conditions for the UDH assay, including using pH 7 and 37℃ as optimal conditions, along with a 1:100 dilution of the UDH enzyme. The second part of this study used the optimized conditions of the two enzyme reactions to test the functionality of the coupled assay. Recombinant GUS enzymes worked well in the coupled assay: ~256U of recombinant GUS enzymes exhibited optimal enzymatic activities with 0.5 mM estrogen glucuronide. The final part of this study will assess the substrate specificity of recombinant GUS enzymes with testosterone and estrogen glucuronides. By investigating sex hormone metabolism by microbial GUS enzymes, I aim to develop a functional biochemical assay to uncover mechanisms influencing sex-specific gut microbiome differentiation and offer insights into potential therapeutic approaches for sex-specific diseases and physiological conditions. Thus, this study contributes to a deeper understanding of the intricate interplay between gut microbiota and host sex-specific physiology, with implications for personalized medicine and targeted interventions.