Glycerophospholipids have been highlighted as the major lipids class affected by trenbolone acetate/estradiol implant administration in bovines. Non-targeted hydrophilic interaction chromatography (HILIC) and reverse phase liquid chromatography (RPLC) coupled to high resolution mass spectrometry have been successfully applied for characterizing lipid profile disruption in serum of implanted bovines. HILIC data pointed towards significant decrease of C22 fatty acids in implanted animals, i.e., C22:3 (p < 0.05), C22:4 (p < 0.05) and C22:6 (p < 0.01), whilst RPLC data confirmed depletion of glycerophospholipids (phosphatidylglycerols, phosphatidylcholine, phosphatidic acid and phosphatidylethanolamine) with C22 fatty acid chains. Using these two complementary methods, complex lipids with the same alkyl chain have been putatively characterized and could further serve to understand the biological mechanism of illicit administration of trenbolone acetate/estradiol implant. These metabolites underpin glycerophospholipid metabolism as potential pathway, which was identified using metabolomic pathway analysis and MetExplore platforms. We hypothesized that implantation of exogenous androgenic and estrogenic steroids induces a depletion of glycerophospholipids with C22 FA chains, by decreasing high-density lipoproteins, the main transporters of glycerophospholipids in plasma.