Defects of mitochondrial fatty acid beta-oxidation are an important group of inherited metabolic disorders in children. Despite improved screening opportunities, diagnosis of these disorders is not often straightforward and requires enzyme analyses. Because therapy is effective in many of these disorders, rapid diagnosis is essential. We report a technique that allows analysis of fatty acid oxidation not only in cultured cells (fibroblasts, myoblasts, and myotubes) but also in fresh muscle homogenate. Fatty acid oxidation analysis was performed by incubating fresh muscle homogenate or harvested cultured cells with stable isotopically labeled palmitate. The intermediates generated were analyzed by tandem mass spectrometry. Results of patients with seven different beta-oxidation disorders were compared with controls. Acylcarnitine intermediates in patient samples could be easily differentiated from the control samples. The acylcarnitine profile of each beta-oxidation defect was compatible with localization of the enzyme defect. Both in patient and control samples, the same pattern of intermediates could be detected in fibroblasts, muscle cells, and fresh muscle homogenate. The procedure described allowed correct diagnosis of all the beta-oxidation defects studied. Utilization of fresh muscle samples reduces the delay in diagnosis related to tissue culture and is useful in diagnostic of patients with neuromuscular phenotype. Measurement of fatty acid oxidation intermediates from myoblasts or myotubes is an additional tool in investigating pathogenetic mechanisms of myopathy in beta-oxidation defects.