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We previously demonstrated that we could hijack the fungal pheromone signaling pathway to provide a living yeast biosensor where peptide biomarkers were recognized by G-protein-coupled receptors and engineered to transcribe a readout. Here, we demonstrated that the protease could be reintroduced to the biosensor to provide a simple mechanism for distinguishing single-amino-acid changes in peptide ligands that, otherwise, would likely be difficult to detect using binding-based assays. We characterized the dose-response curves for five fungal pheromone G-protein-coupled receptors, peptides, and proteases -Saccharomyces cerevisiae , Candida albicans , Schizosaccharomyces pombe , Schizosaccharomyces octosporus , and Schizosaccharomyces japonicus . Alanine scanning was carried out for the most selective of these- S. cerevisiae and C. albicans -with and without the protease. Two peptide variants were discovered, which showed diminished cleavage by the protease (CaPep2A and CaPep2A13A). Those peptides were then distinguished by utilizing the biosensor strains with and without the protease, which selectively cleaved and altered the apparent concentration of peptide required for half-maximal activation for 2 peptides-CaPep and CaPep13A, respectively-by more than one order of magnitude. These results support the hypothesis that the living yeast biosensor with a sequence-specific protease can translate single-amino-acid changes into more than one order of magnitude apparent shift in the concentration of peptide required for half-maximal activation. With further engineering by computational modeling and directed evolution, the biosensor could likely distinguish a wide variety of peptide sequences beyond the alanine scanning carried out here. In the future, we envision incorporating proteases into our living yeast biosensor for use as a point of care diagnostic, a scalable communication language, and other applications., Competing Interests: Competing interests: A provisional patent application no. 63/228,577 was filed 2021 Aug naming V.W.C., T.C., and M. Fantini (Columbia University, USA) as inventors and assigned to The Trustees of Columbia University in the City of New York. V.W.C. holds equity and is Chairman of the Board and President of Celsyntec, Inc. All other authors declare no competing interests.