AIM: Metabolomics applications represent an emerging field where significant efforts are directed. Derivatization consists prerequisite for GC-MS metabolomics analysis.METHODS: Common silylation agents were tested for the derivatization of blood plasma. Optimization of methoxyamination and silylation reactions was performed on a mixture of reference standards, consisting of 46 different metabolites. Stability of derivatized metabolites was tested at 4°C.RESULTS: Optimum results were achieved using N-methyl-N-(trimethylsilyl)trifluoroacetamide. Methoxyamination at room temperature for 24 h followed by 2-h silylation at high temperature lead to efficient derivatization.CONCLUSION: Formation and stability of derivatives among metabolites differ greatly, so derivatization should be studied before application in metabolomics studies.