Metabolomics is thought to be a powerful tool for a comparative display of gene function. However, only a few reports have described the application of metabolomics to functional genomics for the elucidation of unknown or putative genes. The aim of our study is to evaluate the use of metabolomics for functional genomics based on loss-of-function caused by gene deletion selected. In this study, three putative biosynthetic genes (YGR012W, YHR112C, and YML082W) in cysteine metabolism were selected. We first developed a widely targeted metabolomics method using liquid chromatography triple-quadrupole mass spectrometry (LC/MS/MS) to evaluate the effect of single gene deletion. A total of 191 metabolites covering a wide-scope metabolic pathway were analyzed simultaneously through a combination of fast-speed multiple reaction monitoring with positive/negative polarity switching. Using this method, we successfully detected 95 metabolites in yeast cells and characterized the metabolic alteration due to single gene deletion of cysteine biosynthetic enzymes. Our results suggested that metabolomics has the potential not only to determine putative gene function but also to provide new insight into complex metabolic networks.