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An LC–MS/MS assay to quantify vitamin B metabolites has been developed and validated.Assay facilitates the simultaneous determination of concentrations of folates, flavins and B6 metabolites using 50μL of plasma.The method was applied on plasma samples obtained from healthy volunteers and colorectal cancer patients.The method can be used to probe the alteration in the metabolites of one- carbon metabolism in various disease conditions.An analytical method using electrospray ionization and high- performance liquid chromatography/tandem mass spectrometry (LC/ESI–MS/MS) was developed to quantify the vitamin B metabolites found in the folate one-carbon metabolism, using 50μL of human plasma. Analytes in plasma were extracted using protein precipitation after being stabilized in 1% ascorbic acid. The analytes were separated using a Kinetex 2.6μm Pentafluorophenyl (2.1×30mm) column utilizing a gradient mobile phase system of 0.1% formic acid in water and 100% acetonitrile in a 13.2min run. The MS detector run using a positive multiple reaction monitoring with parameters optimized for each analyte’s ion pair. The assay was selective and linear for all analytes at defined dynamic ranges. The recoveries were generally above 80% except for the folate metabolites whose recoveries dipped possibly due to the drying process. The inter-day precision (%coefficient of variation) and accuracy (%calculated concentration of the nominal concentrations) for six replicates of all quality control samples were ≤14% and within 12.2%, respectively. The lower limit of quantification ranged from 0.2 to 3.9nM. No significant instability was observed after repeated freezing and thawing or in processed samples.The LC–MS/MS assay was found applicable for sensitive, accurate and precise quantitation of vitamin B metabolites in plasma of healthy volunteers and colorectal cancer patients.