New chemical and physical properties can be selectively introduced into proteins directly in live cells by genetically incorporating unnatural amino acids. The incorporation efficiency determines how effective such properties can be exploited and was very low in yeast. We developed a new method for efficient expression of orthogonal bacterial tRNA in yeast using polymerase III promoters that are cleaved from primary transcripts. In addition, a yeast strain deficient in nonsense-mediated mRNA decay was generated to prevent rapid degradation of target mRNA containing premature stop codons, which are the most frequently used to encode unnatural amino acids. These new strategies enabled a significant increase in yield of unnatural amino acid containing proteins from tens of micrograms to tens of milligrams per liter in yeast.