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Abstract Improved prodrug‐activating enzymes have the potential to increase the therapeutic efficacy of gene‐directed enzyme prodrug therapy (GDEPT). Yeast cytosine deaminase (yCD) is commonly used to convert the prodrug 5‐fluorocytosine (5‐FC) to the chemotherapeutic 5‐fluorouracil for GDEPT. Mutagenesis studies on yCD aimed at improving its application in GDEPT have been limited to subsets of residues or have sought to improve a single property of the enzyme. We performed comprehensive site‐saturation mutagenesis (CSM) on yCD designed to create all 2,983 possible unique protein mutants with a single amino acid substitution. We identified active variants throughEscherichia coligenetic complementation and screened these mutants, and combinations thereof, for increased ability to sensitizeE. coliand HT1080 fibrosarcoma cells to 5‐FC. Several mutants identified in this study showed increased sensitization ability for bothE. coliand HT1080 cells indicating that CSM is an effective directed evolution tool for identifying unexpectedly beneficial mutations.