The increasing interest in one-carbon metabolism in research filed and industrial field will demand powerful profiling analytical platform enabling the simultaneous determination of multiple components of one-carbon metabolism in large-scale studies. The compounds in one-carbon metabolism are of different chemical structure, various stability and variable polarity. To measure those compounds accurately and precisely with a single method is a true challenge. However, Creative Proteomics has established sensitive, reliable, and accurate HPLC-MS/MS method for quantification of one-carbon metabolism metabolites.