Changes in the lipid metabolism are known to mediate several diseases (e.g. atherosclerosis, coronary artery disease). The potential of lipidomics is increasingly recognized as clinical tool to assess pathomechanisms and biomarkers for several of these diseases. The purpose of this study was first, to evaluate canine lipoprotein separation and second, to characterize the canine lipidome using an untargeted lipidomic approach. After ultracentrifugation or precipitation of plasma and serum, fractions were analyzed by SDS-PAGES, indicating associated apoproteins. No accurate separation of the different lipoproteins was possible. Lipidome analysis revealed a total amount of 561 different lipids from eight of eight categories in plasma and serum; 246 of them were identified in both matrices. The relative mean abundance was different in 11/246 lipids between the two matrices. Prednisolone treatment induced a significant increase of seven and a significant decrease of three lipids in plasma and a significant increase of two and a significant decrease of one lipid in the serum. In conclusion, complete lipoprotein separation is still not possible in dogs, presumably due to their overlapping densities. Untargeted lipidomic analysis revealed marginal differences between lipids found in both, serum and plasma. Prednisolone treatment induced significant changes in the plasma lipidome, which should be evaluated in further studies.