Gene-directed enzyme prodrug therapy is an emerging strategy for cancer treatment based on the delivery of a gene that encodes an enzyme that is able to convert a prodrug into a potent cytotoxin exclusively in target cancer cells. The aim of this study is to determine the capability of bacterial amidohydrolases to activate novel prodrugs and the effect such system has on the viability of eukaryotic cancer cells. HCT116 cancer cell line was subjected to LC-MS/MS proteomic analysis to confirm bacterial YqfB amidohydrolase expression at protein level.