When considering mechanisms of toxicity development to chemical substances, one potentially important mechanism is the selective inhibition of proteins essential for cell growth (target molecules). In this study, to detect the target molecules of chemical substances, we established a method for comprehensively screening for essential proteins that confer resistance against chemical substances via overexpression in yeast. We used budding yeast, a common eukaryotic model organism, to produce yeast strains showing overexpression of different genes encoding essential proteins. This method was used to search for overexpressed genes conferring arsenite resistance in yeast, and as a result, we successfully identified ten types of new genes correlated with arsenite resistance.