We have previously described methodology that makes it possible to genetically encode a wide array of unnatural amino acids in both prokaryotic and eukaryotic organisms. Here, we report the systematic optimization of a Saccharomyces cerevisiae expression system for the production of mutant proteins containing unnatural amino acids. Modifications include significant increases in both the expression levels of the orthogonal Escherichia coli amber suppressor tRNA(CUA) and cognate aminoacyl-tRNA synthetase (aaRS) pair, and expression of the target protein gene using a strong transcriptional promoter, optimized codons and elevated plasmid copy numbers. With this new system, a number of unnatural amino acids, including the photocrosslinkers p-benzoylphenylalanine and p-azidophenylalanine, and the chemically reactive amino acids, p-acetylphenylalanine and p-propargyloxyphenylalanine, were incorporated into human superoxide dismutase (hSOD) in yeast in good yields (maximally approximately 6-8 mg/l of culture in most cases). Mass spectrometric analysis of the hSOD mutants was performed with high dynamic range using multiple reaction monitoring that provided new insights into the factors that control the fidelity of unnatural amino acid incorporation.