Establishing a diagnosis in patients with a suspected mitochondrial disorder is often a challenge. Both knowledge of the clinical spectrum of mitochondrial disorders and the number of identified disease-causing molecular genetic defects are continuously expanding. The diagnostic examination of patients requires a multi-disciplinary clinical and laboratory evaluation in which the biochemical examination of the mitochondrial functional state often plays a central role. In most cases, a muscle biopsy provides the best opportunity to examine mitochondrial function. In addition to activity measurements of individual oxidative phosphorylation enzymes, analysis of mitochondrial respiration, substrate oxidation, and ATP production rates is performed to obtain a detailed picture of the mitochondrial energy-generating system. On the basis of the compilation of clinical, biochemical, and other laboratory test results, candidate genes are selected for molecular genetic testing. In patients in whom an unknown genetic variant is identified, a compatible biochemical phenotype is often required to firmly establish the diagnosis. In addition to the current role of the biochemical analysis in the diagnostic examination of patients with a suspected mitochondria disorder, this report gives a future perspective on the biochemical diagnosis in view of both the expanding genotypes of mitochondrial disorders and the possibilities for high throughput molecular genetic diagnosis.