Mitochondrial oxidative phosphorylation (OXPHOS) is fundamental in all aspects of cellular life in aerobic cells and organisms. It is therefore not surprising that a variety of diseases have been attributed to dysfunction of the OXPHOS enzymes. Assessment of OXPHOS in human samples has proved to be a difficult task over years, even when relying on well-established methods. The complexity and the flexibility of the mitochondrial organization in cells account for a large part in the difficulties encountered in assessing OXPHOS activity. Nevertheless, a careful and detailed analysis of OXPHOS enzyme activity in cells or biopsy samples from patients at risk provides diagnosis of potential OXPHOS deficiency. Problems inherent in the use of human material, mostly the small size of the samples to be analysed, are difficult to resolve. However, cautious handling of these samples permits reasonable confidence to be reached in the interpretation of the data.