High protein (HP) diets are often used as a means to reduce obesity, but their long-term effects remain unclear. In vitro studies suggest the involvement of a subset of oxylipins in the tissue response to HP diets. To examine the role of these bioactive lipids in vivo, normal adult male Sprague Dawley rats were provided isocaloric diets with LP (low protein, 8% protein by weight), NP (normal protein, 14%) or HP (50%) diets for two weeks, and targeted lipidomic analysis of oxylipins in kidney, liver and serum was performed by HPLC-MS/MS. The main group of oxylipins affected by the HP diets was the oxylipins derived from linoleic acid (LA), many of which were elevated in kidney and liver, but reduced in serum of rats provided the HP compared to NP or LP diets. Further studies are needed to elucidate the physiological effects of the changes in these novel oxylipins in response to dietary HP.