Utilizing enzymes as drugs presents a pioneering new feat within drug development. Owing to the catalytic characteristics inherent in enzymes, in contrast to conventional pharmaceuticals, the therapeutic administration necessitates only a minute dosage of the enzyme for an effective treatment.¹ Our design and preparation of novel specific artificial microproteases, establishes a new pathway towards putative treatment of diseases involving pathogenic proteins including cancers, Alzheimer’s, and other chronic diseases.^2,3 While natural proteases consist of hundreds to thousands of amino acids to achieve proteolytic activity, our synthetic enzymes range from 30-50 amino acids. The artificial microproteases were designed by mutational molecular dynamics (MD) using a mixture of L/D-amino acids to avoid tacticity and including non-canonical amino acid building blocks as well. Catalytic residues were incorporated into the folded microproteins at the final stage of design. The synthetic proteases were synthesized using automated SPPS and cyclized by the reaction and/or disulfide bond formation to enhance the structural stability. A one-bead one-compound substrate library was assembled using SPPS and the split/mix method. An intramolecular FRET assay was established by incorporating a 2-aminobenzamide fluorophore at the C-terminus, as well as a 3-nitrotyrosine fluorescence quencher at the N-terminus of the substrate library, to screen for cleavage of active substrates. From these assays, an active artificial Ser-protease was found to have proteolytic activity. Furthermore, one of the Ser-proteases underwent auto-hydrolysis when left in solution for prolonged time. Positive beads showing hydrolysis were isolated to be characterized by MS-MS to determine the substrate specificity and cleavage sites. The Ser-protease is currently being structurally investigated by NMR spectroscopy. The enzyme kinetics are yet to be determined by in-solution assays. The scaffold of the active protease will be adapted to provide specificity towards an Alzheimer’s peptide substrate, and if successful, conjugated to an antibody raised to Alzheimer’s plaques.