Antibody-drug conjugates (ADCs) are a targeted chemotherapeutic currently at the cutting edge of oncology medicine. These hybrid molecules consist of a tumor antigen-specific antibody coupled to a chemotherapeutic small molecule. Through targeted delivery of potent cytotoxins, ADCs exhibit improved therapeutic index and enhanced efficacy relative to traditional chemotherapies and monoclonal antibody therapies. The currently FDA-approved ADCs, Kadcyla (Immunogen/Roche) and Adcetris (Seattle Genetics), are produced by conjugation to surface-exposed lysines, or partial disulfide reduction and conjugation to free cysteines, respectively. These stochastic modes of conjugation lead to heterogeneous drug products with varied numbers of drugs conjugated across several possible sites. As a consequence, the field has limited understanding of the relationships between the site and extent of drug loading and ADC attributes such as efficacy, safety, pharmacokinetics, and immunogenicity. A robust platform for rapid production of ADCs with defined and uniform sites of drug conjugation would enable such studies. We have established a cell-free protein expression system for production of antibody drug conjugates through site-specific incorporation of the optimized non-natural amino acid, para-azidomethyl-l-phenylalanine (pAMF). By using our cell-free protein synthesis platform to directly screen a library of aaRS variants, we have discovered a novel variant of the Methanococcus jannaschii tyrosyl tRNA synthetase (TyrRS), with a high activity and specificity toward pAMF. We demonstrate that site-specific incorporation of pAMF facilitates near complete conjugation of a DBCO-PEG-monomethyl auristatin (DBCO-PEG-MMAF) drug to the tumor-specific, Her2-binding IgG Trastuzumab using strain-promoted azide-alkyne cycloaddition (SPAAC) copper-free click chemistry. The resultant ADCs proved highly potent in in vitro cell cytotoxicity assays.