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INTRODUCTION: Patient-derived skin fibroblasts offer a unique translational model to study molecular mechanisms of multiple human diseases. Metabolomics profiling allows to track changes in a broad range of metabolites and interconnected metabolic pathways that could inform on molecular mechanisms involved in disease development and progression, and on the efficacy of therapeutic interventions. Therefore, it is important to establish standardized protocols for metabolomics analysis in human skin fibroblasts for rigorous and reliable metabolic assessment. OBJECTIVES: We aimed to develop an optimized protocol for concurrent measure of the concentration of amino acids, acylcarnitines, and components of the tricarboxylic acid (TCA) cycle in human skin fibroblasts using gas (GC) and liquid chromatography (LC) coupled with mass spectrometry (MS). METHODS: The suitability of four different methods of cell harvesting on the recovery of amino acids, acylcarnitines, and TCA cycle metabolites was established using GC/MS and LC/MS analytical platforms. For each method, metabolite stability was determined after 48 hours, two weeks and one month of storage at −80°C. RESULTS: Harvesting cells in 80% methanol solution allowed the best recovery and preservation of metabolites. Storage of samples in 80% methanol up to one month at −80°C did not significantly impact metabolite concentrations. CONCLUSION: We developed a robust workflow for metabolomics analysis in human skin fibroblasts suitable for a high-throughput multiplatform analysis. This method allows a direct side-by-side comparison of metabolic changes in samples collected at different time that could be used for studies in large patient cohorts.