A simple LC-MS/MS method was developed and validated for determination of homocysteine and methionine in human plasma. Simultaneous measurement of methionine helps in the differentiation of homocystinuria and some cobalamin disorders (such as cblC and cblD defects) without additional amino acid testing. This assay is sensitive to detect reduced total homocysteine concentrations in sulfocysteinuria and molybdenum cofactor deficiencies.