Site-specific incorporation of non-canonical amino acids (ncAAs) has emerged as a promising tool for synthesizing proteins with novel biological properties. However, the breadth of ncAA incorporation systems is limited by incompatibility between ncAAs and the endogenous protein translation machinery. Specifically, Elongation Factor Tu (EF-Tu), a critical translational component that proofreads and delivers aminoacyl-tRNAs to the ribosome, has been a major barrier for ncAA incorporation. Here we employ reconstructing-evolutionary-adaptive-paths (REAP) analysis to create a site-saturation library based on 48 positions of EF-Tu that potentially modulate its recognition of ncAA-tRNA complexes. Using a novel in vivo antibiotic resistance-based selection assay, we isolate 29 EF-Tu variants can that incorporate p-azido-L-phenylalanine, p-benzoyl-L-phenylalanine or N^ε-L-thiaprolyl-L-lysine via an amber codon. To further evaluate EF-Tu variants that demonstrated substrate-promiscuity in vivo, we constructed an in vitro assessment system based on the PURExpress system. With assistance from flexizyme, we examine the ability of EF-Tu variants to incorporate ncAAs that are incompatible with aminoacyl-tRNA synthetases. These in vitro assays allow us to interrogate EF-Tu variants for their individual abilities to incorporate ncAAs. Collectively, these findings support an evolutionary approach to identify poly-specific or ncAA-specific EF-Tu variants and broaden the scope of ncAA incorporation.