While the pace of discovery of somatic mutations in tumor genomes has rapidly accelerated, deciphering the functional impact of these variants has become rate-limiting. Furthermore, linking somatic variants to specific cancer hallmarks, such as chromosome instability (CIN), is a major challenge in the cancer field. The budding yeast, Saccharomyces cerevisiae, has been utilized to define cellular pathways and catalog a comprehensive list of yeast genes required for the maintenance of chromosome stability (yeast CIN genes). Human orthologs of these yeast genes are candidate CIN genes whose tumor-specific variants may contribute to chromosome instability and tumorigenesis. By ‘humanizing’ a yeast strain using cross-species complementation, yeast can facilitate direct screening of these somatic mutations with the dual benefit that the variants are characterized in the context of the human gene and screened rapidly in a model eukaryote. Here, we outline an experimental approach to identify a list of human genes that can replace their yeast orthologs and complement a loss-of-function phenotype. We report the screening of essential and non-essential yeast CIN deletion mutants for complementation by their human counterparts. Replaceability was scored by examining rescue of growth defects caused by deletion of the essential yeast gene or by assaying for rescue of drug sensitivities for non-essential yeast genes. The resultant list of human gene/ yeast mutant complementation pairs has been used to test the functional consequences of cancer somatic mutations directly in yeast, while also providing a list of complementing genes as a resource to the field.