Mutants of the Angiotensin Converting-Enzyme 2 (ACE2) carboxypeptidase possessing enhanced hydrolytic activity and specificity hold potential to beneficially modulate the Angiotensin receptor (ATR) therapeutic axis with increased efficacy and reduced potential side effects relative to wild type ACE2. In pursuing this goal, we established a yeast display-based liquid chromatography screen that enabled use of directed evolution to identify ACE2 mutants with improved target peptide substrate, Angiotensin-II (Ang-II), activity and specificity relative to Apelin-13, an off-target peptide substrate. Screening yeast-displayed ACE2 active site residue saturation mutant libraries revealed three substitution-tolerant positions that can be mutated to enhance ACE2's activity profile. Double mutant libraries combining substitutions at these positions, M360, T371 and Y510, yielded candidate improved ACE2 mutants that were recombinantly expressed and purified at 1 mg/L yield and > 90% homogeneity. Relative to wild type, the leading mutant, T371L/Y510Ile, has seven-fold increased kcat toward Ang-II and six-fold decreased kcat/Km for Apelin-13 hydrolysis. In single substrate hydrolysis assays featuring physiologically relevant substrate concentrations T371L/Y510Ile hydrolyzes more Ang-II than wild type with concomitant Ang-II:Apelin-13 specificity improvements reaching 30-fold. Additionally, T371L/Y510Ile hydrolyzed Ang-II at rates greater than wild type, with Apelin-13 hydrolysis reductions of up to 80 percent, in multiplex assays containing a mixture of peptides relevant to the ATR therapeutic axis. Our efforts have delivered ATR axis-acting therapeutic candidates with relevance to established and unexplored ACE2 therapeutic applications and demonstrate the feasibility of developing ACE2 variants for use in biomedical contexts unrelated to the ATR axis such as localized activation of peptide-based prodrugs.