Mitochondrial oxidative phosphorylation defects are a common cause of mitochondrial diseases, which are characterized by multiorgan involvement and clinically heterogeneous manifestations. Diagnosis is difficult because of the lack of clinically feasible methods. In this study, mitochondrial complex I-V enzyme activity was measured in 64 patients with suspected mitochondrial disease and 200 healthy controls. Spectrophotometric assay was used for the analysis of mitochondrial complex I-V enzyme activity in peripheral leukocytes. Diagnosis was based on clinical presentation, magnetic resonance imaging (MRI), muscle pathology, and point mutation screening in mitochondrial DNA. The differential diagnosis of aminoacidopathies, organic acidurias, and fatty acid β-oxidation defects was made. Thirty-five patients (54.7%) were diagnosed with Leigh syndrome based on characteristic brain MRI. Complex enzyme activity in controls was found to be stable. A deficiency in the oxidative phosphorylation was found in 29 patients (45.3%). Twenty (31.2%) patients had isolated complex defects, complex I deficiency (n = 2, 3.1%), complex II deficiency (n = 3, 4.7%), complex III deficiency (n = 5, 7.8%), complex IV deficiency (n = 5, 7.8%), and complex V deficiency (n = 5, 7.8%). Nine patients were found to have combined deficiencies, 3 (4.7%) had combined deficiencies of complex I and IV, 2 (3.1%) had combined deficiencies of complex III and V, and 2 (3.1%) had a combined deficiency of complex I and V. In conclusion, the peripheral leukocyte oxidative phosphorylation enzyme activity assay was found to be a reliable method for the diagnosis of mitochondrial diseases.