We present three methods relating to tRNA aminoacylation with non-natural amino acids using an Escherichia coli EF-Tu E215A/D216A mutant that can bind tightly to aa-tRNAs carrying either non-natural or natural amino acids: (i) a method for improving aminoacylation efficiency, (ii) a rapid method for analysing aminoacylation efficiency without the use of radioisotope labelling and (iii) a method for purifying aminoacyl-tRNAs. Although the EF-Tu mutant may be incompatible with some kinds of non-natural amino acids, we confirmed that the EF-Tu mutant could efficiently bind to aa-tRNAs carrying various amino acids (Arg, Ser, O-methyltyrosine, Bodipy FL-aminophenylalanine and 2-acrydonylalanine). These methods may be used for the efficient in vitro synthesis of proteins containing various non-natural amino acids.