Lipidomic studies are gaining importance, as alterations in lipid metabolism have been recognized as being involved in the pathogenesis of several diseases. This study aimed to characterize the plasma lipidome of healthy Beagle dogs using a targeted lipidomic approach and to evaluate alterations in the lipidome after short-term prednisolone and long-term tetracosactide treatment as a model of hypercortisolism. Both treatments led to significant lipidome alterations, however, they were more pronounced and more profound after long-term steroid excess. Lipid load was decreased after short-term and increased after long-term excess. In two detected lipid classes (cholesterol esters and sphingosine-1-phosphate) clear opposite effects were found. After long-term tetracosactide treatment, phosphatidylcholine and lysophosphatidylcholine species with an acyl chain length of <19 carbon atoms and low unsaturation were upregulated, whereas lipid species with a chain length of ³19 carbon atoms and high unsaturation were downregulated. We conclude that evaluation of the whole lipidome is crucial for a complete picture of plasma lipid changes, as the measurement of single lipid classes may obscure relevant alterations. The different findings depending on the duration and type of treatment are a first step in characterizing the influence of steroids on the lipid metabolism; however, the biological significance of these findings clearly has to be evaluated with further studies.