Dihydrolipoamide dehydrogenase (DLD) deficiency (MIM #246900) is a rare autosomal recessive mitochondrial disorder caused by pathogenic variants in the DLD gene, which encodes the E3 subunit common to multiple mitochondrial enzyme complexes, including pyruvate dehydrogenase (PDHc) and α-ketoglutarate dehydrogenase (αKGDHc). Although genotype–phenotype correlations have been described, the precise bioenergetic consequences of DLD dysfunction remain poorly defined. Here, we applied high-resolution respirometry using a novel single-run protocol that allows simultaneous assessment of mitochondrial respiratory capacity and, critically, distinguishing between PDHc- and αKGDHc-linked respiration within the same assay. Fibroblasts from six genetically confirmed DLD-deficient patients with distinct pathogenic variants and clinical severities exhibited a consistent reduction in maximal and complex I-linked respiration. The most severe cases (c.1436A>T; p.D479V) showed combined PDHc and αKGDHc impairment, whereas milder genotypes displayed isolated PDHc dysfunction. This mechanistic distinction likely underlies the variable clinical response to ketogenic therapy, which depends on intact αKGDHc function. Analysis of the mitochondrial mass and mtDNA copy number revealed no global reduction, indicating intrinsic enzymatic dysfunction as the primary defect. Collectively, this study defines a reproducible bioenergetic signature of DLD deficiency and introduces an integrated one-run diagnostic strategy for delineating enzyme-specific mitochondrial defects, providing a framework for mechanistic and therapeutic investigations.