Many enzyme families contain both catalytically active and inactive members. Although these inactive enzyme-homologs (pseudoenzymes) share significant similarity with their active counterparts, key active site modifications seem to suggest a loss of traditional catalytic activity. A key technique for elucidating the function of non-essential genes, including those producing pseudoenzyme products, is the genome-wide synthetic lethal screen. Biochemical pathways typically involve multiple key gene components and many of these key gene players provide redundancy in the event that other genes experience deleterious mutations. The synthetic lethal screen is used to identify novel mutant yeast strains whose survival is dependent on a particular gene of interest. Colonies that contain a putative synthetically lethal gene pair are kept alive and identified by harboring a color-reporting plasmid that supplements the deletion of the endogenous gene of interest. Here we demonstrate the construction of this integral pSLS1 plasmid harboring a gene of interest that was amplified and isolated from a wild-type yeast strain using a high-fidelity polymerase chain reaction. Additionally, we demonstrate the validation of pSLS1 plasmid constructs and the determination of gene insert orientation using restriction endonucleases and agarose gel electrophoresis. Finally, we demonstrate the preparation of target yeast strains using drug resistant deletion cassettes and subsequent validation using low-fidelity polymerase chain reactions.