Biosensors play an important role in medical diagnostics, industrial biotechnology, and environmental monitoring. A key challenge in biosensor design is developing complementary ligand binding domains which bind to the small molecule in a sandwich like fashion, causing fused biosensor output domains to co-localise and transduce a signal. We lay the foundation of designing an in vivo system utilising yeast mating for tackling the difficult task of generating novel pairs of complementary binding domains termed as Simultaneous Yeast Display (SYD). This system relies on strains of yeast that have their native sexual agglutination ability knocked out such that mating of haploid cells to form a diploid is dependent upon interactions between heterologous expressed surface proteins. While this system has been used previously to characterise and select for proteinprotein interactions, its use in selecting protein-ligand-protein interactions has not been published. We present results showing that surface display of known ligand binding proteins causes an increase in mating efficiency upon introduction of the ligand in liquid culture. In the future, this technique can be further used to screen yeast display libraries of randomised peptides or proteins to discover novel complementary binding domains with high sensitivity and selectivity for a multitude of ligands.