Mitochondrial oxidative phosphorylation (OXPHOS) is responsible for producing most of the adenosine triphosphate required by eukaryotic cells. Lymphocytes make up the majority of the peripheral blood mononuclear cells. Peripheral blood mononuclear cells are readily obtainable, providing an ideal sample to monitor systemic changes and understand molecular signaling mechanisms in disease processes. Mitochondrial energy metabolism of lymphocyte has been used to screen for OXPHOS disorders. While there are increasing studies of lymphocyte OXPHOS, few studies examined activity of electron transport chain of lymphocyte mitochondria. We present an optimal protocol to harvest fresh peripheral blood mononuclear cells from human whole blood, determine integrated mitochondrial function, and analyze electron transport chain complex activity. Analyzing integrated mitochondrial function using OXPHOS provides data to uncover defects in the transport of substrates into the mitochondria, generation of reducing equivalents, the electron transport chain, and coupling to the production of adenosine triphosphate. The optimal conditions to harvest peripheral blood mononuclear cells were using blood anticoagulated with ethylenediaminetetraacetic acid, processed utilizing Lymphoprep™, and washed in phosphate buffered saline, all at room temperature. Using isolated peripheral blood mononuclear cells, integrated mitochondrial function and the activities of electron transport chain were determined.