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If early diagnosis is not made, patients with peroxisomal disorders rapidly progress to sudden death, physical defect or mental retardation resulted in storage of the toxic material into the brain. Therefore, it is necessary to develop the analytical method for rapid screening and/or correct confirmation diagnosis. The method utilizes [2H(9)]pipecolic acid as internal standard. The formation of trimethylsilyl derivative (TMS) of the carboxylic functional group was performed by adding MSTFA. And then 5 microL of methyl orange was added until the color of methyl orange was to yellow. Consecutively, the trifluoroacyl (TFA)-derivative of the -NH functional group was produced by adding MBTFA. GC-MS was set with specific ions (m/z 282, m/z 297) of the TMSTFA derivative of pipecolic acid for selected ion monitoring. The linearity of pipecolic acid in pooled plasma spots showed 0.9999 in the range of 10-150 ng investigated. The precision and accuracy was within S.D. of 5% (RSD, within 5%) for intra-day and inter-day assay. Normal control value has been determined in plasma spots of infant and adults aged 0-30 (including newborn). The utility of the method was demonstrated by quantifying various concentration of fortified pipecolic acid on a filter plasma spot. The new method was simple with just two step derivatisation, time and labor saving without SPE or liquid-liquid extraction, and convenience of delivery owing to the use of filter paper. The described method could be used for routine analysis, monitoring, and clinical diagnostic application of peroxisomal disorders on dietary therapy.