The genetic code of living cells has been reprogrammed to enable the site-specific incorporation of hundreds of non-canonical amino acids (ncAAs) into proteins, and the synthesis of non-canonical polymers and macrocyclic peptides. Current methods for engineering orthogonal aminoacyl-tRNA synthetases (aaRSs) rely on translational readouts, requiring the monomers to be ribosomal substrates. This creates an evolutionary deadlock, limiting the scope of translation to α-t-amino acids and related hydroxy acids. To break this deadlock, we developed tRNA display, a method that enables the direct, rapid, and scalable selection of orthogonal synthetases that selectively acylate their cognate orthogonal tRNAs with ncMs, regardless of whether the ncMs are ribosomal substrates. Using tRNA display, we selected orthogonal aaRSs that specifically acylate their cognate tRNAs with eight ncMs, including β-amino acids, α,α-disubstituted amino acids, and β-hydroxy acids. We further demonstrated the site-specific incorporation of β-amino acids and α,α-disubstituted amino acids into proteins in Escherichia coli, expanding the chemical scope of the genetic code. This work paves the way for the genetic encoding and site-specific incorporation of a broader range of monomers, potentially enabling the creation of protease-resistant proteins and new drug-like molecules.The genetic code of living cells has been reprogrammed to enable the site-specific incorporation of hundreds of non-canonical amino acids (ncAAs) into proteins, and the synthesis of non-canonical polymers and macrocyclic peptides. Current methods for engineering orthogonal aminoacyl-tRNA synthetases (aaRSs) rely on translational readouts, requiring the monomers to be ribosomal substrates. This creates an evolutionary deadlock, limiting the scope of translation to α-t-amino acids and related hydroxy acids. To break this deadlock, we developed tRNA display, a method that enables the direct, rapid, and scalable selection of orthogonal synthetases that selectively acylate their cognate orthogonal tRNAs with ncMs, regardless of whether the ncMs are ribosomal substrates. Using tRNA display, we selected orthogonal aaRSs that specifically acylate their cognate tRNAs with eight ncMs, including β-amino acids, α,α-disubstituted amino acids, and β-hydroxy acids. We further demonstrated the site-specific incorporation of β-amino acids and α,α-disubstituted amino acids into proteins in Escherichia coli, expanding the chemical scope of the genetic code. This work paves the way for the genetic encoding and site-specific incorporation of a broader range of monomers, potentially enabling the creation of protease-resistant proteins and new drug-like molecules.