Knowledge about synthetic lethality can be applied to enhance the efficacy of anti-cancer therapies in individual patients harboring genetic alterations in their cancer that specifically render it vulnerable. We investigated the potential for high-resolution phenomic analysis in yeast to predict such genetic vulnerabilities by systematic, comprehensive, and quantitative assessment of drug-gene interaction for gemcitabine and cytarabine, substrates of deoxycytidine kinase that have similar molecular structures yet distinct anti-tumor efficacy. Human deoxycytidine kinase (dCK) was conditionally expressed in the S. cerevisiae genomic library of knockout and knockdown (YKO/KD) strains, to globally and quantitatively characterize differential drug-gene interaction for gemcitabine and cytarabine. Pathway enrichment analysis revealed that autophagy, histone modification, chromatin remodeling, and apoptosis-related processes influence gemcitabine specifically, while drug-gene interaction specific to cytarabine was less enriched in Gene Ontology. Processes having influence over both drugs were DNA repair and integrity checkpoints and vesicle transport and fusion. Non-GO-enriched genes were also informative. Yeast phenomic and cancer cell line pharmacogenomics data were integrated to identify yeast-human homologs with correlated differential gene expression and drug-efficacy, thus providing a unique resource to predict whether differential gene expression observed in cancer genetic profiles are causal in tumor-specific responses to cytotoxic agents.