Background: Nitric oxide (NO) plays an important role in endothelial homeostasis. Asymmetric dimethyl arginine (ADMA), L-N monomethyl arginine (L-NMMA) and symmetric dimethyl arginine (SDMA), which are derivatives of methylarginine, directly or indirectly reduce NO production. Therefore, these metabolites are an important risk factor for various diseases, including cardiovascular diseases. Numerous methods have been developed for the measurement of methylarginine derivatives, but various difficulties have been encountered. This study aimed to develop a reliable, fast and cost-effective method for the analysis and measurement of methylarginine derivatives (ADMA, SDMA, L-NMMA) and related metabolites (arginine, citrulline, homoarginine, ornithine), and to validate this method according to Clinical and Laboratory Standards Institute (CLSI) protocols. Methods: For the analysis of ADMA, SDMA, L-NMMA, arginine, homoarginine, citrulline, ornithine, 200 µl of serum were precipitated with methanol, and subsequently derivatized with a butanol solution containing 5% acetyl chloride. Butyl derivatives were separated using a C18 reverse phase column with a 5 min run time. Detection of analytes was achieved by utilising the specific fragmentation patterns identified through tandem mass spectrometry. Results: The method was linear for ADMA, SDMA, L-NMMA, ornithine, arginine, homoarginine and citrulline in the ranges of 0.023–6.0, 0.021–5.5, 0.019–5.0, 0.015–250, 0.015–250, 0.019–5 and 0.015–250 µM, respectively. The inter-assay CV% values for all analytes was less than 9.8%. Conclusions: Data obtained from method validation studies shows that the developed method is highly sensitive, precise and accurate. Short analysis time, cost-effectiveness, and multiplexed analysis of these metabolites, with the same pretreatment steps, are the main advantages of the method.