Protein and peptide therapeutics are of growing importance as medical treatments but can frequently induce an immune response. This work describes the combination of complementary approaches to map the potential immunogenic regions of the yeast Rhodosporidium toruloides phenylalanine ammonia-lyase (PAL, EC 18.104.22.168) and to engineer the protein as a human therapeutic agent for the treatment of phenylketonuria (PKU), an inherited metabolic disorder. The identification of B and T cell epitopes on the PAL protein was performed by computational predictions based on the antigenicity and hydrophilicity of proteins, as well as by experimental epitope mapping using a PepSpots peptide array (Jerini AG). Human T cell epitope mapping was performed by applying the computational EpiMatrix algorithm (EpiVax, Inc.) for MHC Class I and Class II associated T cell epitopes on PAL, which predicts which sequences are associated with binding to several different HLA alleles, a requirement for antigen presentation and subsequent primary immune response. By chemical modification through PEGylation of surface lysine residues, it is possible to cover the immunogenic regions of a protein. To evaluate this strategy, we used mass spectrometry to determine which of the immunogenic epitopes are covered by the covalent PEGylation modification strategy. This approach has allowed us to determine whether additional lysines are needed in specific residue locations, or whether certain lysine residues can be removed in order to accomplish complete molecular coverage of the therapeutic enzyme.