Lemon juice is used to enhance the flavor of dishes prepared with fish, which is more prone to oxidation than other flesh food. This study evaluated the impact of treating sardine fillets (Sardina pilchardus) with Tahiti lemon juice and submit them to different domestic cooking methods (air fryer, grill, and deep-fat fryer), regarding the oxidation of unsaturated fatty acids and cholesterol. The juice extract was characterized by its antioxidant capacity and bioactive constituents by UHPLC-MS. Cooking caused the degradation of polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic (EPA, C20:5) and docosahexaenoic acids (DHA, C22:6), and increased the cholesterol oxides level. However, for most treatments, degradation of PUFAs and formation of cholesterol oxides were more pronounced in samples treated with juice. Lemon juice induced cholesterol oxidation, mainly in air-fried sardines where the total content of cholesterol oxides increased from 700 ± 4 (control) to 1333 ± 5 μg/g (10 % lemon juice, in dry basis). Thus, the overall data suggest that potential antioxidant compounds may have degraded during heating and the acid environment may have induced oxidative reactions.