Mitochondrial oxidative phosphorylation (OXPHOS) dysfunction is implicated in a growing spectrum of diseases, from neurodegeneration to cancer. Where tissues or transformed cells are available, respirometry and enzymology allow a sophisticated analysis of OXPHOS with modest-cost equipment. The isolation of organelle fractions is also invaluable for determining association of proteins of interest. Here we revisit and consolidate methods to measure whole cell mitochondrial ATP synthesis, respiration, isolation of mitochondria from cultured cells and tissues, and OXPHOS enzymology. We also explain common pitfalls, guide optimisation of the methods for new users, and provide full laboratory protocols in Supplementary materials. (C) 2014 Elsevier B.V. and Mitochondria Research Society. All rights reserved.