Mitochondrial disorders (MD) comprise a group of heterogeneous clinical disorders for which non-invasive diagnosis remains a challenge. Two protein biomarkers have so far emerged for MD detection, FGF-21 and GDF-15, but the identification of additional biomarkers capable of improving their diagnostic accuracy is highly relevant. Previous studies identified Gelsolin as a regulator of cell survival adaptations triggered by mitochondrial defects. Gelsolin presents a circulating plasma isoform (pGSN), whose altered levels could be a hallmark of mitochondrial dysfunction. Therefore, we investigated the diagnostic performance of pGSN for MD relative to FGF-21 and GDF-15. Using ELISA assays, we quantified plasma levels of pGSN, FGF-21, and GDF-15 in three age- and gender-matched adult cohorts: 60 genetically diagnosed MD patients, 56 healthy donors, and 41 patients with unrelated neuromuscular pathologies (non-MD). Clinical variables and biomarkers’ plasma levels were compared between groups. Discrimination ability was calculated using the area under the ROC curve (AUC). Optimal cut-offs and the following diagnostic parameters were determined: sensitivity, specificity, positive and negative predictive values, positive and negative likelihood ratios, and efficiency. Comprehensive statistical analyses revealed significant discrimination ability for the three biomarkers to classify between MD and healthy individuals, with the best diagnostic performance for the GDF-15/pGSN combination. pGSN and GDF-15 preferentially discriminated between MD and non-MD patients under 50 years, whereas FGF-21 best classified older subjects. Conclusion: pGSN improves the diagnosis accuracy for MD provided by FGF-21 and GDF-15.