Lactate, pyruvate, beta-hydroxybutyrate, and acetoacetate are intermediary metabolites normally occurring in blood. Their relative concentrations are an expression of nutritional balance in energy metabolism. The simultaneous determination of these four analytes is of special interest in the understanding of energy homeostasis. Rapid, precise routine measurement of these four analytes is basic for the first orientation of the diagnosis of inborn errors of intermediary metabolism, especially those affecting fuel homeostasis. We describe an adaptation of the Cobas Fara II centrifugal analyser of some previously reported methods using the supernatant of a unique deproteinized blood sample for the determination of the four analytes. Reference values are calculated for a pediatric population. The difficulties involved in the diagnosis and follow-up of mitochondrial diseases in children require the standardization of analytical procedures for the correct interpretation of the results.